CN102766689B

CN102766689B - Sequencing method for increasing sequencing reading length

Info

Publication number: CN102766689B
Application number: CN201210232676.4A
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-04-17
Filing date: 2012-07-06
Publication date: 2015-07-22
Anticipated expiration: 2032-07-06
Also published as: CN102766689A

Abstract

The invention relates to the field of biological gene engineering, and provides a sequencing method for increasing sequencing reading length. The sequencing method provided by the invention comprises the following steps of: firstly, combining a first anchor primer on a first connector of a nucleic acid fragment to be measured, and reading sequence information of M nucleotide behind the extending tail end of the first anchor primer by virtue of a connection sequencing method; recognizing corresponding enzyme digestion recognition sites by virtue of incision enzyme, partially or fully cutting the nucleotide of which the sequence information is read, connecting a new second connector with a fragment to be sequenced in enzyme digestion recovery, combining a second anchor primer on the second connector in an anchoring manner, and connecting a new fluorescent probe with the extending tail end of the second anchor primer, wherein the new fluorescent probe extends forwards to read nucleotide sequence information; and circulating the operations to obtain the nucleotide sequence information required by the nucleic acid fragment to be measured, thereby increasing the sequencing reading length by virtue of a enzyme digestion sequencing extending method.

Description

A kind of sequence measurement increasing order-checking and read to grow

Technical field

The present invention relates to biological gene engineering field, more particularly, relate to a kind of sequence measurement increasing order-checking and read to grow.

Background technology

At present, the gold standard detecting gene order sudden change is sequence measurement, and commonly sanger sequencing and Manganic pyrophosphate complex initiation method (Pyrosequencing), wherein Pyrosequencing is applicable to high throughput analysis.Pyrosequencing is when checking order, and the magnetic bead being connected with sequenced fragments is fixed in the aperture of etching optical fiber slide (PTP plate).Because aperture comparatively large (55 μm × 44 μm) is therefore for magnetic bead position when making order-checking immobilizes, filling in aperture is needed to contain the mixture of multiple protein to ensure order-checking and to adopt carrying out smoothly of figure, add the use of luciferase, these factors cause the cost of Pyrosequencing high.

For making order-checking cost reduce, prior art adopts connection sequencing to replace Pyrosequencing to check order.Existing a kind of sequencing that connects utilizes endonuclease digestion extension to check order, as shown in Figure 1, the step of the method comprises: double chain oligonucleotide first joint that (1), utilization cut recognition site containing enzyme is connected with nucleic acid fragment, obtains determined nucleic acid fragment; (2), to identify that restriction enzyme that enzyme cuts recognition site carries out enzyme to nucleic acid fragment to be measured and cuts, the double-stranded products that wherein chain contains protruding terminus is obtained; (3), double-stranded products connects one group of corresponding specific position contain fluorescently-labeled double-strand second joint, obtain connecting product; Nucleotide sequence information corresponding to this specific position is obtained by detecting the fluorescent signal connecting product; Wherein, double-strand second joint contains protruding terminus; Double-strand second joint contains enzyme and cuts recognition site; Cut the Nucleotide number between recognition site and restriction enzyme site according to enzyme, what a or several Nucleotide described protruding terminus precalculate; (4) the connection product, in separating step (3), obtains separated product; (5), utilize can in identification step (3) cut recognition site with enzyme enzyme enzyme carried out to separated product cut, obtain a pack section of cutting recognition site containing enzyme; (6), the operation of repeating step (3) to (5), until record all nucleotide sequences that determined nucleic acid fragment can be surveyed; Wherein during last repetitive operation, can omit step (5).

In above-mentioned connection sequencing, utilize containing fluorescently-labeled stranded oligonucleotide linkers as detection probes, the change of cutting recognition site position with institute's band enzyme on joint realizes and controls the extension propelling of order-checking position.If use the method to detect nucleotide sequence, can because stranded oligonucleotide linkers Nucleotide number and the Nucleotide number between the restriction enzyme enzyme recognition site used and restriction enzyme site limit, make to detect the nucleotide sequence obtained can only equal at most enzyme and cut Nucleotide number between recognition site and restriction enzyme site, it reads long critical constraints system, is unfavorable for the examination and analysb of nucleic acid sequence information.

Therefore need a kind of increase order-checking newly to read long sequence measurement, the length of reading when detecting nucleotide sequence can be made to increase, be convenient to the examination and analysb of nucleic acid sequence information.

Summary of the invention

The object of the present invention is to provide a kind of increase order-checking newly to read long sequence measurement, be intended to solve in prior art utilize connect to read when sequencing detects long too short and be unfavorable for the problem of the examination and analysb of nucleic acid sequence information.

In order to realize goal of the invention, a kind of sequence measurement reading length that checks order that increases comprises the following steps:

A. the first anchor primer is incorporated on the first joint in determined nucleic acid fragment;

B. at the fluorescent probe of the first anchor primer extending end respectively connecting band different positions mark, and detect the fluorescent signal of corresponding connection product, obtain the sequence information of M Nucleotide after the first anchor primer extending end;

C. utilizing restriction endonuclease by having obtained the nucleotide segment of sequence information in step B or having excised completely, obtaining the digestion products containing treating sequenced fragments;

D. digestion products connects the second joint and obtains new determined nucleic acid fragment, is incorporated into by the second anchor primer on the second joint of new determined nucleic acid fragment;

E. at the fluorescent probe of the second anchor primer extending end respectively connecting band different positions mark, and detect the fluorescent signal of corresponding connection product, obtain the sequence information of N number of Nucleotide after the second anchor primer extending end;

F. change restriction endonuclease, joint, anchor primer and fluorescent probe, the product of previous step is carried out that enzyme is cut, joint connects, anchor primer combines, fluorescent probe connects and fluorescent signal detects;

G. repeating step F, until obtain nucleotide sequence information required in determined nucleic acid fragment;

Wherein, M, N are positive integer; Described determined nucleic acid fragment contains nutrition guide gene order, conventional medicine related gene sequence or tumor susceptibility gene sequence.

Wherein, the fragment of determined nucleic acid described in steps A is fixed on surface of solid phase carriers.

Wherein, step can also be comprised before steps A:

A0. utilize solid phase carrier to increase to source nucleic acid, be fixed the determined nucleic acid fragment in surface of solid phase carriers.

Further, described steps A 0 comprises the following steps:

A01. by the source cDNA chip that is used for increasing in surface of solid phase carriers, obtain the solid phase carrier of surface containing at least one nucleic acid fragment;

A02. primer is incorporated into the primer binding site on surface of solid phase carriers, is fixed the amplification vector of primer;

A03. the source nucleic acid on amplification vector is increased, be fixed the determined nucleic acid fragment in surface of solid phase carriers.

Wherein, primer described in steps A 02 comprises upstream primer for increasing to described source nucleic acid and/or downstream primer, described upstream primer holds the complementary nucleotide sequence combined, and described downstream primer is the nucleotide sequence identical with source nucleic acid 3 ' terminal sequence.

Wherein, the amplification described in steps A 03 is unit molecule amplification.

Wherein, in described steps A 02, primer is incorporated into the mode of surface of solid phase carriers and is: the group that primer and surface of solid phase carriers carry carries out pairing and is connected, and realizes directly combining; Or carry out pairing by the group that the group that connexon carries carries with primer and surface of solid phase carriers respectively and be connected, realize indirectly combining.

Further, the mode that described pairing connects adopts at least one in biotin-avidin/Streptavidin, nanometer gold/iodacetyl-sulfydryl, amino-aldehyde radical/carboxyl/isothiocyano, acrylamide-silylation/polyacrylamide.

Wherein, the first anchor primer described in steps A contains at least one enzyme and cuts recognition site.

Wherein, described step C can comprise the following steps:

C1. by the fluorescent probe that connects in step B and the first anchor primer wash-out, reset the first anchor primer and carry out chain extension, forming double chain acid molecule with determined nucleic acid fragment;

C2. restriction endonuclease by identification first anchor primer with enzyme cut recognition site and carry out enzyme and cut, by having obtained the nucleotide segment of sequence information in step B or having excised completely, obtained the digestion products containing treating sequenced fragments.

Wherein, step C can comprise the following steps:

C1 '. connect the joint three of double-strand at the other end of the first anchor primer, this joint three cuts recognition site containing at least one enzyme;

C2 '. utilize restriction endonuclease identification joint three with enzyme cut recognition site, by having obtained the nucleotide segment of sequence information in step B or having excised completely, obtained the digestion products containing treating sequenced fragments.

In above-mentioned either a program, described first anchor primer contains at least one specificity residue and/or one end is closed.

Wherein, described step B comprises the following steps:

B1. at the fluorescent probe of the first anchor primer extending end connecting band specific position mark containing specificity residue, detect the fluorescent signal of corresponding connection product, obtain the nucleotide sequence information of correspondence position;

B2. with specificity cutting agent cleavage specificity residue, by the fluorescent probe that connects in previous step and the first anchor primer wash-out, the first anchor primer is reset;

B3. repeat the operation of being with the connection of fluorescent probe of specific position mark and corresponding fluorescent signal to detect at the first anchor primer extending end, obtain the sequence information of M Nucleotide after the first anchor primer extending end.

Wherein, described nutrition guide gene comprise Green Tea Extract remove gene, drinking gene, anti-smoking lesioned gene, detoxification ability gene, DNA-repair gene, folic acid and vitamins B receptivity gene, vitamins D receptivity gene, cholesterol metabolic ability gene, low-density lipoprotein metabolic capacity gene, calcium and phosphorus receptivity gene, melanin deposition and metabolic gene and dairy digestive and absorption base because of at least one.

Wherein, described conventional medicine genes involved comprises at least one of CYP1A2, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2D6, ABCB1, ADRB1, ADRB2, CACNA1C, NPPA, OPRM1 and VKORC1.

Wherein, described tumor susceptibility gene comprises at least one in tumor susceptibility gene, cardiovascular tumor susceptibility gene, metabolic system tumor susceptibility gene and immunity system tumor susceptibility gene.

Further, described tumor susceptibility gene comprises at least one in breast cancer susceptibility gene, lung cancer tumor susceptibility gene, susceptibility gene of colorectal cancer, nasopharyngeal carcinoma susceptibility genes, stomach cancer susceptible genes, liver cancer susceptibility, carcinoma of the pancreas tumor susceptibility gene, skin carcinoma tumor susceptibility gene, ovarian cancer susceptible gene, prostate cancer tumor susceptibility gene and leukemia tumor susceptibility gene; Described cardiovascular tumor susceptibility gene comprises at least one in atherosclerosis susceptible gene, coronary heart disease tumor susceptibility gene, essential hypertension tumor susceptibility gene, parkinsonism tumor susceptibility gene and senile dementia tumor susceptibility gene; Described metabolic system tumor susceptibility gene comprises at least one in insulin-dependent diabetes mellitus (IDDM) tumor susceptibility gene, type II diabetes tumor susceptibility gene, fatty liver tumor susceptibility gene and hypoglycemia tumor susceptibility gene; Immunity system tumor susceptibility gene comprises at least one in osteoporosis susceptible gene, anaemia tumor susceptibility gene, systemic inflammatory response syndrome tumor susceptibility gene and psoriasis predisposing genes.

As from the foregoing, long sequence measurement is read in increase order-checking of the present invention, restriction endonuclease is utilized determined nucleic acid fragment to have obtained the nucleotide segment of sequence information or to have excised completely, and then connect new joint, the anchor primer that grappling is new also carries out the connection of fluorescent probe and the detection of fluorescent signal, extend forward and read nucleotide sequence, thus add order-checking and read long.

Accompanying drawing explanation

Fig. 1 is a kind of connection sequencing schematic diagram utilizing endonuclease digestion to extend order-checking in prior art.

Fig. 2 increases order-checking to read long sequence measurement schema in one embodiment of the invention.

Fig. 3 utilizes solid phase carrier to increase to obtain the method flow diagram of determined nucleic acid fragment in one embodiment of the invention.

Fig. 4 is the structural representation of an anchor used in one embodiment of the invention.

Fig. 5 is the method flow diagram utilizing the anchor shown in Fig. 4 to obtain the sequence information of M Nucleotide after its extending end in one embodiment of the invention.

Fig. 6 is the structural representation of an anchor in the present invention's embodiment.

Fig. 7 is the method flow diagram utilizing the anchor shown in Fig. 6 to obtain the sequence information of M Nucleotide after its extending end in the present invention's embodiment.

Fig. 8 is the structural representation of an anchor in another embodiment of the present invention.

Fig. 9 is the method flow diagram utilizing the anchor shown in Fig. 8 to obtain M position nucleotide sequence information after its extending end in the present invention's embodiment.

Figure 10 obtains the method flow diagram containing the digestion products treating sequenced fragments in one embodiment of the invention.

Figure 11 obtains the method flow diagram containing the digestion products treating sequenced fragments in another embodiment of the present invention.

Figure 12 is the method flow diagram of the sequence information obtaining N number of Nucleotide after the 2nd anchor extending end in one embodiment of the invention.

Figure 13 is the method schematic diagram of the sequence information obtaining N number of Nucleotide after the 2nd anchor extending end in another embodiment of the present invention.

Figure 14 utilizes enzyme to cut to extend the sequence measurement schematic diagram that length is read in order-checking increase order-checking in one embodiment of the invention.

Embodiment

In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.

The invention provides a kind of sequence measurement increasing order-checking and read to grow, comprise the following steps: first utilize the first anchor primer (anchor) the first joint grappling in determined nucleic acid fragment to be combined, the fluorescent probe utilizing different positions to mark is connected with the extending end of an anchor, and detect the fluorescent signal of corresponding connection product, the sequence information of M Nucleotide after an anchor extending end in conventional medicine genes involved determined nucleic acid fragment is read according to fluorescent signal; Utilize restriction endonuclease to carry out enzyme to cut, by the nucleotide sequence portion read before or excise completely, retain containing wait check order nucleotide sequence wait check order nucleic acid fragment, and make it be connected with new joint, and then the anchor primer that grappling is new (anchor), then by the fluorescent probe of the extending end connecting band different positions mark of new anchor, the sequence information of N number of Nucleotide after reading the extending end of new anchor; Complete one by above-mentioned steps and detect circulation, the step that the connection of endonuclease digestion excision described before then repeating, joint replacing connection, new anchor grappling, new fluorescent probe and fluorescent signal detect, circulation is read, thus obtains the nucleotide sequence information in determined nucleic acid fragment.Utilize the increase described in the present invention to check order and read long sequence measurement, what can increase order-checking reads length.

It should be noted that, the sequence measurement reading length that checks order that increases of the present invention can be used for detecting nutrition guide gene order, conventional medicine associated genotype sequence and tumor susceptibility gene sequence.

Described nutrition guide gene refers to copying, transcribes, undergo mutation in accurate translation process after gene that the metabolism of nutritive substance is had an impact, wherein the metabolic process of nutritive substance comprises digestion, transhipment, absorbs and excretion.Nutrition guide gene can set up personalized nutritional recipe in order to instruct, and regulates dietetic alimentation.Nutrition guide gene is detected, individual Genetic uniqueness can be understood, and by adjustment of diet, formulate personalized nutritious recipe, be conducive to nutritional supplementation and the equilibrium of individuation, realize the reasonable absorption of individuation nutrition.

The individual difference of drug reaction is phenomenon extremely general clinically, and the reason producing this species diversity has many, is mainly divided into non-heritable agency and hereditary factors, and both combinations can cause different patient to the reaction appearance amount of same medicine and qualitative difference.Non-heritable agency comprises the many factors of sex, age, body weight, disease condition; Inherited genetic factors, the gene pleiomorphism of the also i.e. metabolism of conventional medicine, release, intracellular signaling and acceptor causes the changing function of its proteins encoded.

Described conventional medicine genes involved refers to encode to conventional medicine metabolism in vivo, release, intracellular signaling and receptor protein relevant gene or allelotrope.Conventional medicine genes involved is detected, the nucleic acid sequence information of concrete conventional medicine genes involved can be obtained.Gained nucleic acid sequence information, i.e. hereditary factors, combine with non-heritable agency, can draw the difference of medicine drug effect between Different Individual, realize personalized medicine.The defining method of non-genetic factor includes but not limited to further molecular biology test, clinical trial, clinical observation and comprehensive analysis etc.So utilize method of the present invention to detect conventional medicine genes involved, what obtain is only the nucleic acid sequence information of conventional medicine genes involved, and can not in order to judge the curative effect of medicine.

Described tumor susceptibility gene refers to have certain association with human body specific characterization, can disclose human body genetic constitution to the gene of healthy pros and cons and disease susceptibility situation or allelotrope.The tumor susceptibility gene of complex disease is detected, concrete nucleic acid sequence information can be obtained.On this basis, can in conjunction with the statistical study of follow-up further molecular biology test, clinical trial, clinical observation and integrated data, set up disease model, thus realize the susceptibility of earlier evaluations disease and take corresponding preventive measures to reduce the occurrence probability of disease.Method of the present invention is utilized to detect to tumor susceptibility gene the nucleic acid sequence information that the detected result obtained is tumor susceptibility gene, whether not ill in order to directly to judge.

The present invention is when detecting nutrition guide gene, conventional medicine genes involved or tumor susceptibility gene, and its initial sample carries out processing or detecting for the tissue departing from human body or animal body, body fluid or excremental sample.These samples include but not limited to blood, oral epithelium scrapes sampling, saliva, urine, paraffin-embedded tissue and puncturing tissue.

Wherein, anchor primer of the present invention is anchor, and the joint referring in determined nucleic acid fragment carries out the single stranded oligonucleotide that grappling is combined; The extending end of described anchor, referring to that can be used in continuation connects and the anchor end carrying out nucleotide chain extension, can be the 5 ' end of anchor, also can be the 3 ' end of anchor.

Fluorescent probe described in the present invention is divided into different type of sets, with the different IPs nucleotide sequence of the corresponding same specific position of fluorescent mark different in set type, and the specific position difference that the fluorescent probe fluorescent mark of different set type is corresponding.In each ligation, add the fluorescent probe of same set type, according to gathered fluorescent signal, the nucleotide sequence information that this group fluorescent probe mark specific position is corresponding can be read.

The fluorescent mark kind of fluorescent probe described in the present invention is not limit, include but not limited to one, two kinds, four kinds, or more.

When fluorescent mark only has one, in order to distinguish the different bases on same position, to the base on same position, 4 ligations and fluorescent signal detection need be repeated at the extending end of anchor primer, in each ligation, fluorescent probe is a certain base (A, G, C or T) on corresponding a certain specific position and containing fluorescently-labeled probe.In order to realize the detection of the base to x position, then need the detection repeating 4 × x time ligation and fluorescent probe.

When fluorescent mark has two kinds, in order to distinguish the different bases on same position, to the base on same position, 2 ligations and fluorescent signal detection need be repeated at the extending end of anchor primer, in each ligation, fluorescent probe is certain two kinds of base (A, G, C or T) on corresponding a certain specific position and containing fluorescently-labeled probe.In order to realize the detection of the base to x position, then need the detection repeating 2 × x time ligation and fluorescent probe.

When fluorescent mark has four kinds, in order to distinguish the different bases on same position, to the base on same position, 1 ligation and fluorescent signal detection need be carried out at the extending end of anchor primer, in each ligation, fluorescent probe is certain four kinds of base (A, G, C or T) on corresponding a certain specific position and containing fluorescently-labeled probe.In order to realize the detection of the base to x position, then need the detection repeating x ligation and fluorescent probe.

When fluorescently-labeled kind is more, sequential detection can be carried out with reference to such scheme.Preferably, fluorescently-labeled kind can be divided exactly by 4 or can divide exactly 4, to simplify the design of fluorescent probe and follow-up test experience.

In addition, in the present invention, when the extending end of anchor connects fluorescent probe, mainly contain two kinds of ways of realization, before the difference between them is mainly to connect fluorescent probe at every turn, whether again grappling anchor: if not grappling anchor again, then after the connection and corresponding fluorescent signal that complete each fluorescent probe detect, by the fluorescent probe excision connected on product, retain anchor, then connect new fluorescent probe, then carry out fluorescent signal detection; If grappling anchor again, then after the connection and corresponding fluorescent signal that complete each fluorescent probe detect, whole connection product (connector of fluorescent probe and anchor) is removed, then grappling anchor again, connect new fluorescent probe, and gather the fluorescent signal connecting product further.

Fig. 2 shows in one embodiment of the invention increases the sequence measurement flow process that length is read in order-checking, and the method comprises the following steps:

S1. an anchor is incorporated on the first joint in determined nucleic acid fragment;

S2. at the fluorescent probe of an anchor extending end respectively connecting band different positions mark, and detect the fluorescent signal of corresponding connection product, obtain the sequence information of M Nucleotide after an anchor extending end;

S3. utilizing restriction endonuclease by having obtained the nucleotide segment of sequence information in step S2 or having excised completely, obtaining the digestion products containing treating sequenced fragments;

S4. digestion products connects the second joint and obtains new determined nucleic acid fragment, is incorporated into by the 2nd anchor on the second joint of new determined nucleic acid fragment;

S5. at the fluorescent probe of the 2nd anchor extending end respectively connecting band different positions mark, and detect the fluorescent signal of corresponding connection product, obtain the sequence information of N number of Nucleotide after the 2nd anchor extending end;

S6. change restriction endonuclease, joint, anchor and fluorescent probe, the product of previous step is carried out that enzyme is cut, joint connects, anchor combines, fluorescent probe connects and fluorescent signal detects;

S7. repeating step S6, until obtain nucleotide sequence information required in determined nucleic acid fragment;

Long sequence measurement is read in increase order-checking described in the present invention, it is advantageous that, when carrying out sequencing reaction and detecting nucleotide sequence, restriction endonuclease is utilized determined nucleic acid fragment to have read the nucleotide segment of sequence information or to have excised completely, and then containing wait check order nucleotide sequence nucleic acid fragment on connect new joint, the anchor that grappling is new also carries out the connection of new fluorescent probe and the detection of corresponding fluorescent signal, realization extends forward reads more nucleotide sequence, thus the order-checking adding utilization connection sequencing detection nucleotide sequence is read long.

For the technical scheme described in the present invention, it should be noted that, described in step S1, an anchor is according to base pair complementarity principle, and the first joint in determined nucleic acid fragment carries out the anchor primer that grappling is combined, single stranded oligonucleotide, for connecting fluorescent probe in step s 2.In addition, an anchor according to specific needs, can also carry out different designs.

In one embodiment of the invention, an anchor carries out grappling with first joint of determined nucleic acid fragment one end by base pair complementarity and is combined.In the program, an anchor can not carry out sealing treatment, to reduce the synthesis cost of an anchor; Also can carry out sealing treatment in one end of an anchor, avoid an anchor to occur from connecting each other, ensureing that an anchor is connected with the orientation of fluorescent probe.

Being closed one end of one anchor, can be the 3 ' end of an anchor, also can be 5 ' end, both can the direction of control linkage through sealing treatment, can avoid again being interconnected between an anchor.In an embodiment of this step, 3 ' of one anchor end is closed, the method closed includes but not limited to two deoxidation, ammoxidation, amidation, and the 3 ' end that an anchor is closed cannot continue to connect, and namely the connection of an anchor can only occur in 5 ' end; And in another embodiment of this step, 5 ' of anchor end is closed, the method closed includes but not limited to dephosphorylation or amidation, and namely the connection of an anchor can only occur in 3 ' end.

In another embodiment of the invention, one anchor contains at least one specificity residue, described specificity residue refer to itself or its with the residue that cut by specificity of chemical bond energy, it can make the nucleotide fragments at its place due to cut and be easier to wash-out.Material for cleavage specificity residue is called specificity cutting agent.Described specificity residue include but not limited to deoxyuridine acid (deoxy-Uracil, dU), Hypoxanthine deoxyriboside (deoxy Inosine, dI), Nucleotide containing phosphorothioate bond and nickase enzyme cut recognition site.Its corresponding specificity cutting agent includes but not limited to uracil-DNA glycosylase (Uracil-DNA Glycocasylase, UDG enzyme), E. coli endonuclease V, compound containing Ag, Hg, Cu, Mn, Zn or Cd ion and nickase.Described nickase, refers to and can identify that the enzyme in double chain acid molecule entrained by a nucleotide chain cuts recognition site, and on the nucleotide chain cutting recognition site containing this enzyme, carry out the Type II restriction enzyme that enzyme cuts formation otch; Nickase of the present invention, includes but not limited to: Nt.Alw I restriction endonuclease, Nt.BsmA I restriction endonuclease, Nt.BspQ I restriction endonuclease, Nt.BstNB I restriction endonuclease.It is more convenient that the one anchor design of the present embodiment can make in follow-up wash-out Renewal process.

In another embodiment of the invention, one anchor contains at least one enzyme and cuts recognition site, this enzyme cut recognition site can be used directly in partially or completely to excise in step S2 the nucleotide sequence that read, whole invention technical scheme steps is simplified, easy and simple to handle.

Stating the first joint in step S1 is one section of known array on determined nucleic acid fragment one end, being positioned at 3 ' end or the 5 ' end of determined nucleic acid fragment, being attached to determined nucleic acid fragment for making an anchor grappling.This first joint can be obtain sample to be checked order after to carry out in sequencing library building process designed, designed synthesis and connect, also can be that order-checking sample copy body contains treating of obtaining.

Described nutrition guide gene, refer to copying, transcribe, undergo mutation in accurate translation process after gene that the metabolism of nutritive substance is had an impact, nutrition guide gene can set up personalized nutritional recipe in order to instruct, and regulates dietetic alimentation.Described nutrition guide gene include but not limited to Green Tea Extract remove gene, drinking gene, anti-smoking lesioned gene, detoxification ability gene, DNA-repair gene, folic acid and vitamins B receptivity gene, vitamins D receptivity gene, cholesterol metabolic ability gene, low-density lipoprotein metabolic capacity gene, calcium and phosphorus receptivity gene, melanin deposition and metabolic gene and dairy digestive and absorption base because of.

Wherein, described Green Tea Extract is removed gene and is included but not limited to CAT, CYBA, NOS3, SOD3 and PON1; Described drinking gene includes but not limited to ADH2, ADH3, ALDH2 and CYP2E1; Described anti-smoking lesioned gene and detoxification ability gene include but not limited to CYP1A1; Described DNA-repair gene includes but not limited to PARP1, XRCC1 and ERCC2; Described folic acid and vitamins B receptivity gene include but not limited to MTHFR, MTR and MTRR; Described vitamins D receptivity gene includes but not limited to VDR; Described cholesterol metabolic ability gene and low-density lipoprotein metabolic capacity gene include but not limited to ABCB1, LPL, APOE and APOB; Described calcium and phosphorus receptivity gene include but not limited to VDR; Described melanin deposition and metabolic gene include but not limited to MC1R, OCA2 and TYRP1; Described dairy digestive and absorption base are because including but not limited to LCT and MCM6.

Wherein, described nutrition guide gene includes but not limited to ABCB1, ADH2, ADH3, ALDH2, APOB, APOE, CAT, CYBA, CYP1A1, CYP2E1, ERCC2, LCT, LPL, MC1R, MCM6, MTHFR, MTR, MTRR, NOS3, OCA2, PARP1, PON1, SOD3, TYRP1, VDR and XRCC1.

Wherein, described tumor susceptibility gene includes but not limited to tumor susceptibility gene, cardiovascular tumor susceptibility gene, metabolic system tumor susceptibility gene and immunity system tumor susceptibility gene.

Described tumor susceptibility gene includes but not limited to breast cancer susceptibility gene, lung cancer tumor susceptibility gene, susceptibility gene of colorectal cancer, nasopharyngeal carcinoma susceptibility genes, stomach cancer susceptible genes, liver cancer susceptibility, carcinoma of the pancreas tumor susceptibility gene, skin carcinoma tumor susceptibility gene, ovarian cancer susceptible gene, prostate cancer tumor susceptibility gene and leukemia tumor susceptibility gene.

Described breast cancer susceptibility gene includes but not limited to FGFR2, GSTP1, IL-1 β, MTHFR and GSTM1; Described lung cancer tumor susceptibility gene includes but not limited to APE1, CASP7, CASP8, CASP9, CHEK2, COX-2, CYP1A1, CYP2E1, ERCC1, ERCC2, ERCC6, Exo1, GSTP1, Hmlh1, IL-1 β, MDM2, MTHFR, OGG1, P73, TERT, TGFB1, TP53, TP63 and XRCC1.

Described susceptibility gene of colorectal cancer includes but not limited to MMP2, SMAD7, ADH2, ALDH2, CYP1A2, MMP-1, MTHFR, TP53, VEGF, COX-2, DNMT3B, hMLH1, LOC727677, MMP9, MTRR and TGF-β 1.

Described nasopharyngeal carcinoma susceptibility genes includes but not limited to IL-2, MDM2, HLA-A, HLA-B/C, HLA-F, MDS1-EVI1, CDNK2A/2B, TNFRSF19, HCG9, GABBR1 and ITGA9.

Described stomach cancer susceptible genes includes but not limited to EGF, ALDH2, MTHFR, p53, IL-8, IL-10, PSCA, TNFA, PLCE1, CYP1A1, XRCC1, CYP2E1, TGFB1, CDH1, MDM2 and VEGF.

Described liver cancer susceptibility includes but not limited to IL1-B, TNF-α, EGF, TGF-β 1, DEPDC5, MICA and KIF1B.

Described carcinoma of the pancreas tumor susceptibility gene includes but not limited to MTHFR, COX-2, FasL, CASP8, THSD7B, ARL4C, LTF, FOXQ1, PARK2, FAM91A1, RNF5P1, RNF43, BICD1, NDFIP2, PRPSAP2, MYO1D and LOC.

Described cardiovascular tumor susceptibility gene includes but not limited to atherosclerosis susceptible gene, coronary heart disease tumor susceptibility gene, essential hypertension tumor susceptibility gene, parkinsonism tumor susceptibility gene and senile dementia tumor susceptibility gene.

Described atherosclerosis susceptible gene includes but not limited to ALOX5AP, ApoE, LDLR, HUMARA, DCC and Rb.

Described coronary heart disease tumor susceptibility gene includes but not limited to MEF2A, PDGF, FGF, EGF, VEGF, COX-1 and T-PA.

Described essential hypertension tumor susceptibility gene includes but not limited to AGT, ACE, AT1R, CYP11B2, G-β 3, eNOS, RnBO, LDLR and LL.

Described parkinsonism tumor susceptibility gene includes but not limited to SNCA, LRRK2, PINK1, UCH-L1 and Parkin.

Described senile dementia tumor susceptibility gene includes but not limited to APP, PS-1, PS-2, ApoE, ACE, CH25H, CST3, CHRNB2 and SORL1.

Described metabolic system tumor susceptibility gene includes but not limited to insulin-dependent diabetes mellitus (IDDM) tumor susceptibility gene, type II diabetes tumor susceptibility gene, fatty liver tumor susceptibility gene and hypoglycemia tumor susceptibility gene.

Described immunity system tumor susceptibility gene includes but not limited to osteoporosis susceptible gene, anaemia tumor susceptibility gene, systemic inflammatory response syndrome tumor susceptibility gene and psoriasis predisposing genes.

Described conventional medicine genes involved, refers to encode to conventional medicine metabolism in vivo, release, intracellular signaling and receptor protein relevant gene or allelotrope.

Wherein, described conventional medicine includes but not limited to: the cough medicine morphine monomethyl ether in respiratory system relevant diseases medicine and Dextromethorphane Hbr, anti-asthmatic theophylline, anti-infective voriconazole, itraconazole, dapsone, metronidazole, sulfamethoxazole and Rifampin in anti-pathogenic microorganism medicine, anti-HIV agent viracept see nelfinaivr, antimalarial drug Quinine Sulphate Di HC and chloroguanide, treatment Rezulin Glyburide, Glipizide, glimepiride and nateglinide in endocrine system relevant diseases medicine, hormone medicine Progesterone and dexamethasone, antiandrogen medicine flutamide, anodyne methadone in nervous system disease agent, fentanyl, morphine and tramadol hydrochloride, maincenter skeletal muscular relaxant tizanidine, antipyretic-antalgic anti-inflammatory agent diclofenac, Tenoxicam, Ibuprofen BP/EP (Ibuprofen), Naproxen Base, piroxicam (piroxicam), flurbiprofen, indomethacin, lornoxicam, Celebrex, acetylsalicylic acid and paracetamol, thymoleptic nortriptyline, imipramine (Tofranil), chlorimipramine, fluoxetine Hydrochloride, Sertraline, paroxetine, venlafaxine, tomoxetine, citalopram, moclobemide, clobazam, Trimipramine, etizolam, trazodone, fluvoxamine and duloxetine, anti-neuropathy medicine trilafon, haloperidol, risperidone, Aripiprazole, chlorpromazine and leoponex, antiepileptic drug Phenytoin Sodium Salt, stable (diazepam), phenylethyl barbituric acid, Sodium Valproate and Carbamzepine, control migraine agent zolmitriptan, antigout drug colchicine, gastrointestinal upset medicine in Digestive tract relevant diseases medicine, antiemetic dronabinol, cisapride and ondansetron, azoles is drawn in antiulcer drug omeprazole, lansoprazole and day care, beta receptor blocking agent bufuralol in recycle system relevant diseases medicine, metoprolol and bisoprolol, antihypertensive drug carvedilol, Debrisoquine, losartan, valsartan and irbesartan, anti-arrhythmic Quinidine, encainide, sparteine, flecainide, Propafenone, Aprindine, mexiletine and lignocaine, anti-anginal drug Parker former times woods (perhexiline), anticoagulant warfarin, blood lipid regulation medicine Simvastatin, lovastatin and fluvastatin, calcium antagonists felodipine, nifedipine, nitrendipine, amlodipine, nimodipine, verapamil and Odizem, antiplatelet drug clopidogrel, and immunosuppressive drug ciclosporin.

Described conventional medicine genes involved includes but not limited to CYP1A2, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2D6, ABCB1, ADRB1, ADRB2, CACNA1C, NPPA, OPRM1 and VKORC1.

Wherein, respiratory system relevant diseases pharmaceutical relevant gene includes but not limited to CYP1A2, CYP2D6.Anti-pathogenic microorganism medicine genes involved includes but not limited to CYP2C19, CYP3A4, CYP2C9 and CYP3A5.Endocrine system relevant diseases pharmaceutical relevant gene includes but not limited to CYP2C19, CYP2C9, CYP3A4 and CYP1A2.Nervous system disease agent genes involved includes but not limited to CYP2D6, CYP3A4, CYP2C9, CYP1A2, CYP2C19, ABCB1 and OPRM1.Digestive tract relevant diseases pharmaceutical relevant gene includes but not limited to CYP2C9, CYP3A4, CYP2D6, CYP1A2 and CYP2C19.Recycle system relevant diseases pharmaceutical relevant gene includes but not limited to CYP2D6, CYP2C9, CYP2C19, CYP1A2, CYP3A4, CYP3A5, ABCB1, VKORC1, CACNA1C, ADRB1, ADRB2 and NPPA.

Determined nucleic acid fragment described in step S1, can be any one in DNA, RNA or cDNA, and at least joint is contained at its two ends, for making to operate facility during order-checking, preferably will wherein be connected with solid phase carrier by an end connector, the described solid phase carrier for connecting can be unlike material and difform rigidity material, and its material includes but not limited to: glass, silicon, pottery, plastics and metal; Its shape includes but not limited to: flaggy shape, plate shaped, disc and spherical; For solid phase carrier, the preferred magnetic bead of the present invention and slide.

The described determined nucleic acid fragment containing joint, its source can be the determined nucleic acid fragment connecting joint obtained, and can be directly used in order-checking; Also can be by obtaining structure sequencing library.If obtain determined nucleic acid fragment by building sequencing library, before step S1, then need the operation carrying out sequencing library structure, the present invention preferably adopts following steps to carry out: S0. utilizes solid phase carrier to increase to source nucleic acid, is fixed the determined nucleic acid fragment in surface of solid phase carriers.

Operation in described step S0 as shown in Figure 3, specifically comprises the following steps:

S01. by the source cDNA chip that is used for increasing in surface of solid phase carriers, obtain the solid phase carrier of surface containing at least one nucleic acid fragment;

S02. primer is incorporated into the primer binding site on surface of solid phase carriers, is fixed the amplification vector of primer;

S03. the source nucleic acid on amplification vector is increased, be fixed the determined nucleic acid fragment in surface of solid phase carriers.

The technical program is used to carry out sequencing library structure, the source nucleic acid being used for increasing and the primer that is used for amplification of nucleic acid fragment are incorporated into surface of solid phase carriers simultaneously, amplified production can be fixed on surface of solid phase carriers, improving the utilization ratio of solid phase carrier when increasing, improving the amplified production binding capacity of surface of solid phase carriers; Utilize the method amplified production obtained that increases to check order, because the raising of the amplified production binding capacity of surface of solid phase carriers, further can strengthen the detection signal of order-checking, reduce the requirement to detecting instrument.

For the technical program, it should be noted that, described source nucleic acid is single stranded nucleic acid molecule, can be DNA, RNA or cDNA, can be directly from organism, tissue or cell, extract the nucleic acid molecule obtained, also can be carry out the product after pcr amplification to the direct nucleic acid molecule obtained that extracts from organism, tissue or cell.

Wherein, solid phase carrier described in the technical program, can be made up of unlike material, its material can adopt in glass, silica gel, pottery, plastics and metal any one, and the surface of solid phase carrier is without particular requirement, solid phase carrier preferably containing smooth-flat-surface, the particular type of solid phase carrier can be solid phase carrier conventional in prior art, includes but not limited to plastic bead, granulated glass sphere, slide, magnetic bead and nm gold particles.Preferably adopt magnetic bead as solid phase carrier in the present invention, convenient to make amplified reaction terminate the separation and purification of rear amplified production.Above-mentioned is the present invention's some specific embodiments for solid phase carrier, not in order to limit the scope of the invention.

In a specific embodiments of the present invention, directly before amplification, fragmentation is carried out to the source nucleic acid that will increase, then the nucleic acid fragment two ends utilizing joint and fragmentation to obtain are connected, and surface of solid phase carriers carries out corresponding moditied processing, then the nucleic acid fragment after joint is fixed on surface of solid phase carriers.

In another specific embodiments of the present invention, the source nucleic acid for increasing is arranged in clinical blood sample to be measured at first.First Streptavidin modification is carried out to surface of solid phase carriers, be then connected through the capture probe of biotin modification, obtain the solid phase carrier containing capture probe.Solid phase carrier containing capture probe is directly mixed with clinical blood sample to be measured, therefrom carries out catching of source nucleic acid.After catching end, utilize centrifugation, the solid phase carrier of the fixing active nucleic acid in surface can be obtained.

In another specific embodiments of the present invention, the source nucleic acid for increasing is arranged in the saliva of clinical disease patient.For obtaining the source nucleic acid for increasing, first corresponding primer is utilized to carry out general PCR, the source nucleic acid obtained from the saliva of patient is amplified, then Separation and Recovery is carried out with gel electrophoresis, reclaim product to be connected with biotinylation joint again, and the magnetic bead that solid phase carrier adopts Streptavidin to modify, both mix combination, can obtain the magnetic bead that surface is fixed with at least one nucleic acid fragment.

Above-mentioned is only some specific embodiments of the source nucleic acid source that in the present invention, surface of solid phase carriers is fixed, not in order to limit the scope of the invention.

Primer described in step S02, being the nucleotide sequence that the source nucleic acid for fixing surface of solid phase carriers increases, comprising at least one in upstream primer and downstream primer.Described upstream primer holds the complementary nucleotide sequence combined; Described downstream primer is the nucleotide sequence identical with 3 ' terminal sequence of source nucleic acid.

In one particular embodiment of the present invention, upstream primer or downstream primer are incorporated into the primer binding site of surface of solid phase carriers, the scheme being simultaneously fixed on surface of solid phase carriers with upstream primer and downstream primer is compared, and can reduce the kind of reagent while realizing goal of the invention; In another specific embodiment of the present invention, upstream primer, downstream primer are incorporated into the primer binding site of surface of solid phase carriers according to certain ratio simultaneously, the speed increased can be accelerated while realizing goal of the invention.Wherein the blending ratio of upstream primer and downstream primer is variable as required, be preferably 1:2 to 2:1 ratio between, be more preferably 1:1.Above-mentioned is the present invention's some embodiments used for the blending ratio between upstream primer and downstream primer, not in order to limit the scope of the invention.

Primer binding site described in step S02, refers to the surface of solid phase carriers site combined with primer.

Amplification vector described in step S02, refer to that surface is fixed with at least one nucleic acid fragment, and be also combined with the solid phase carrier of primer simultaneously, now nucleic acid fragment and primer are fixed on surface of solid phase carriers simultaneously, can directly apply to amplification.

When described in the present invention, primer is incorporated into surface of solid phase carriers, do not get rid of primer and be combined with nucleic acid fragments complementary simultaneously yet, this does not affect the realization of goal of the invention of the present invention.

Primer described in step S02 is incorporated into surface of solid phase carriers and various ways can be adopted to realize.In a specific embodiments of the present invention, primer is connected by carrying out pairing with the group that surface of solid phase carriers carries, and realizes directly combining, and can simplify the operation.In another specific embodiments of the present invention, primer is by being connected with one of them group that connexon carries, another group carried by connexon is again carried out pairing with the group that surface of solid phase carriers carries and is connected, thus realizes primer and be connected with the indirect of solid phase carrier; In another specific embodiment of the present embodiment, connexon is the existence being similar to resin structure, use this connexon except can realize primer and solid phase carrier indirect be connected except, the primer quantity that surface of solid phase carriers combines can also be improved further.

Wherein, described connexon is for connecting primer and solid phase carrier.Described connexon can adopt multiple compounds, includes but not limited to: alkane, single stranded nucleotide acid molecule or comprise the compound of polymer part.

The mode that above-mentioned pairing connects is varied, can adopt any one in biotin-avidin/Streptavidin, nanometer gold/iodacetyl-sulfydryl, amino-aldehyde radical/carboxyl/isothiocyano, acrylamide-silylation/polyacrylamide.

In a specific embodiments of the present invention, primer contains biotin labeling, and solid phase carrier itself has been modified through Streptavidin, and therefore both are directly connected by the pairing between vitamin H with Streptavidin, realizes directly connecting.

In another specific embodiments of the present invention, surface of solid phase carriers contains amido modified, and primer is through carboxyl modified, and both carry out pairing by amino-carboxyl and connect, and realize directly connecting.

In another specific embodiments of the present invention, adopt poly-compounds if resin is as connexon, by amino respectively with primer with carboxyl and the aldehyde radical that carries of surface of solid phase carriers carry out pairing and be connected, realize the indirect combination of primer and surface of solid phase carriers.

In another specific embodiments of the present invention, using alkane molecule as connexon, containing amino and carboxyl on it, therefore can carry out pairing with the solid phase carrier of carboxylated primer and surface amination and be connected, realize the indirect combination of primer and surface of solid phase carriers.

Above-mentioned is only some embodiments that in the present invention, primer is incorporated into surface of solid phase carriers, not in order to limit the scope of the invention.

The combination of primer and surface of solid phase carriers in step S02, can be consistent with the combination of source nucleic acid and surface of solid phase carriers in step S01, also can take different combination.In one embodiment of the invention, primer and source nucleic acid all adopt the mode be connected of directly matching with surface of solid phase carriers.Further, when adopting identical combination mode, source nucleic acid and primer can also adopt identical or different groups to match and realize being connected with direct or indirect pairing of solid phase carrier.In a specific embodiment of the present embodiment, surface of solid phase carriers contains Streptavidin to be modified, and source nucleic acid and primer all contain biotin modification, and source nucleic acid and primer are all fixed on surface of solid phase carriers by the effect of the affine vitamin H of strepto-; In another specific embodiment of the present embodiment, surface of solid phase carriers contains amido modified, and source nucleic acid contains carboxyl modified, and primer contains aldehyde group modified, and source nucleic acid and primer are matched by different groups and realize being connected with direct pairing of solid phase carrier; In another specific embodiment of the present embodiment, surface of solid phase carriers contains amido modified, and source nucleic acid contains carboxyl modified, primer contains isothiocyano to be modified, and source nucleic acid and primer are matched by different groups and realize being connected with direct pairing of solid phase carrier; In another specific embodiment of the present embodiment, solid phase carrier contains amino and Streptavidin after different moditied processing, and source nucleic acid contains carboxyl modified, primer contains biotin modification, and source nucleic acid and primer are matched by different groups and realize being connected with direct pairing of solid phase carrier; In another specific embodiment of the present embodiment, surface of solid phase carriers contains resin embedding, and source nucleic acid and primer match respectively at identical or different group entrained on resin, thus the mode indirectly connected all is adopted to realize the connection with solid phase carrier.

In another embodiment of the invention, source nucleic acid and primer adopt different fixed forms to be combined with surface of solid phase carriers.In a specific embodiment of the present embodiment, surface of solid phase carriers contains Streptavidin to be modified, source nucleic acid realizes being connected with the indirect pairing of solid phase carrier by capture probe, and primer is matched with the direct of solid phase carrier be connected by the effect realization of the affine vitamin H of strepto-, both are connected with solid phase carrier by different combinations; In another specific embodiment of the present embodiment, surface of solid phase carriers contains amido modified, source nucleic acid utilizes carboxyl modified to realize being connected with direct pairing of solid phase carrier, primer by amino with on connexon with aldehyde radical match and be connected, and then realize being connected with the indirect of solid phase carrier by aldehyde radical on connexon, thus realize source nucleic acid and be connected with solid phase carrier by different combinations from primer.

Above-mentioned embodiment and specific embodiment are only some embodiments that in the present invention, source nucleic acid and primer are combined with surface of solid phase carriers, not in order to limit the scope of the invention.

Wherein, the primer in step S02 can be the specificity amplification primer for the nutrition guide gene that increases, conventional medicine genes involved or tumor susceptibility gene.

In step S03, utilize amplification vector to increase to source nucleic acid, refer to and utilize certain method (methods as chemistry, enzymatic or other types) that the copy number of source nucleic acid is increased or cause the signal of source nucleic acid existence to increase.When the technical program utilizes amplification vector to increase, except adding necessary amplifing reagent, also add the primer of a small amount of free state in order to accelerate the speed of startup and the amplification of increasing.The free state primer amount added can adjust accordingly according to the difference of primer kind fixing on solid phase carrier and amount.

In one embodiment of the invention, what surface of solid phase carriers was fixing is upstream primer, and in a specific embodiment, the free state primer added is downstream primer; In another specific embodiment, for enabling amplification rate accelerate, while adding a large amount of free state downstream primer, also add a small amount of upstream primer.

In another embodiment of the invention, what surface of solid phase carriers was fixing is downstream primer, and in a specific embodiment, the free state primer added is upstream primer; In another specific embodiment, for enabling amplification rate accelerate, while adding a large amount of free state upstream primer, also add a small amount of downstream primer.

In another specific embodiments of the present invention, upstream primer and downstream primer are incorporated into surface of solid phase carriers simultaneously, and in a specific embodiment, the free state primer added is wherein a kind of in upstream primer or downstream primer; In another specific embodiment, add upstream primer and the downstream primer of free state during amplification, to accelerate the speed of amplification, and the amount of upstream primer and downstream primer is good with 1:1, also can with other ratios simultaneously.

The present invention can adopt multiple amplification mode, include but not limited to common polymerase chain reaction (PCR) and ligase chain reaction (LCR), strand displacement amplification (SDA), transcriptive intermediate amplification, copy and rolling circle amplification (RCA) based on the amplification (NASBA) of nucleotide sequence, Q-Beta, preferred emulsion PCR(EPCR), bridge-type PCR, wherein bridge-type PCR can be divided into again aqueous phase bridge-type PCR and emulsion bridge-type PCR two kinds.

Unit molecule amplification of the present invention, refer to source nucleic acid, spatially isolate (but these source nucleic acid still belong to same reaction system on the whole) with the form of denier (or even individual molecule), the amplification to source nucleic acid is realized in respective space, obtain the homogeneous amplified production that increases, the signal of the amplified production obtained after increasing in order to lifting.

Wherein, EPCR described in the present invention utilizes the separate space that in emulsion system, each drop is formed, independent amplification reaction is carried out to the source nucleic acid on amplification vector, in order to generate the unit molecule amplification technique of amplified production homogeneous in a large number, its roughly operation steps be: the aqueous solution comprising all reacted constituents of PCR is injected into high speed rotating mineral oil surface, the aqueous solution forms the numerous little water droplet wrapped up by mineral oil instantaneously.These little water droplets just constitute independently PCR reaction compartment.Ideally, each little water droplet only containing an amplification vector, includes enough other amplifing reagents (comprising archaeal dna polymerase, dNTP etc.).After EPCR reaction, surface of solid phase carriers is just fixed with the source nucleic acid amplification product in the same source of copy huge amount.EPCR concrete steps can reference: BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions, Frank Diehl, Meng Li, Yiping He, nature methods, Vol.3, No.7, July 2006.

Bridge-type PCR described in the present invention utilizes the upstream primer that is fixed on solid phase carrier or downstream primer and source nucleic acid to form bridge-like structure to increase, thus obtains the unit molecule amplification technique of a large amount of homology, homogeneous amplified production.The ultimate principle of described bridge-type PCR is, the primer of bridge-type PCR is fixed on solid phase carrier, in PCR process, pcr amplification product can be fixed on solid phase carrier, and pcr amplification product can match with the Primers complementary on solid phase carrier, become bridge like, then complementary pairing primer with the amplified production of its Cheng Qiao for template increases.By controlling the amount that adds of original template, after bridge-type PCR react, the form of amplified production with cluster bunch on solid phase carrier exists, and the amplified production of every cluster is the DNA profiling amplified production with originating.The key distinction of aqueous phase bridge-type PCR and emulsion bridge-type PCR is, emulsion bridge-type PCR carries out bridge-type PCR in the separate space of isolating in emulsion system, has the characteristic of aqueous phase bridge-type PCR and EPCR simultaneously.Can with reference to Publication about Document about the concrete principle of bridge-type PCR and embodiment: CN20061009879.X, US6227604 and Dual primer emulsion PCR for next generation DNA sequencing, Ming Yan Xu et al Benchmarks Vol.48 No.5,2010.

In one embodiment of the invention, utilize PCR method to increase to source nucleic acid, this method is simple to operate, but shortcoming to be to need source nucleic acid more.

In another embodiment of the invention, utilize the method for RCA to increase to source nucleic acid, the advantage of this embodiment is to form unit molecule amplification, can obtain amplified production homogeneous in a large number after amplification terminates.

In a preferred embodiment of the invention, utilize EPCR to increase to the source nucleic acid that amplification vector surface is fixing, unit molecule amplification can be realized, complete amplification with minute quantity or even single source nucleic acid, and obtain amplified production homogeneous in a large number.

In another preferred embodiment of the present invention, utilize bridge-type PCR to increase to the source nucleic acid that amplification vector surface is fixing, unit molecule amplification can be realized equally, complete amplification with minute quantity or even single source nucleic acid, obtain amplified production homogeneous in a large number.

In a specific embodiment in the present embodiment, aqueous phase bridge-type PCR is utilized to increase to the source nucleic acid that surface of solid phase carriers is fixing; And in another specific embodiment in the present embodiment, utilize the amplification that emulsion bridge-type PCR realizes the source nucleic acid that surface of solid phase carriers is fixed.

Wherein, the amplified production described in the present invention, refers to after amplified reaction, and surface is fixed with the solid phase carrier of the nucleotide sequence obtained that increases in a large number.

EPCR and bridge-type PCR is utilized to carry out unit molecule amplification in above-mentioned preferred embodiment, with only have primer or only active cDNA chip in solid phase carrier technology compared with, can avoid owing to lacking wherein a kind of and cannot increase, thus the efficiency of solid phase carrier for increasing can be improved, reduce the usage quantity of solid phase carrier, reduce costs.

Above-mentioned is only some embodiments of the source nucleic acid fixed for the surface of solid phase carriers that increases in the present invention, not in order to limit the scope of the invention.

After step S03 increases, in the amplified production obtained, comprise the solid phase carrier that surface bonding has nucleic acid fragment to be measured, and the impurity that other unreacteds are complete, therefore, before carrying out subsequent operations, need the purifying carried out amplified production to reclaim.The object that purifying reclaims is surface bonding had the solid phase carrier of nucleic acid fragment to be measured and magazins' layout to purify out, can use common method of the prior art, include but not limited to centrifugation purification, column separating purification.

In step S2, the extending end of a described anchor, refers to and can continue to connect after an anchor and carry out the end of nucleotide chain extension.

According to the different designs structure of an anchor described in step S1, step S2 can adopt different implementations.

In one embodiment of the invention, an anchor contains specificity residue, and in a specific embodiment in the present embodiment, as shown in Figure 4, X shown in figure is A, G, C or T to the structure of an anchor, and Y represents specificity residue, and n is positive integer.In addition, the number of Y and the position changeable of Y in X in figure, when there is multiple Y, might not be exist with the form be connected shown in Fig. 4 between Y and Y, can be dispersed in the different positions of an anchor respectively.An above-mentioned anchor can to make in step S2 the wash-out of fluorescent probe and the realization of replacing easier.

Utilize an anchor of this structure to realize the method for step S2 as shown in Figure 5, comprise the following steps:

S21. at the fluorescent probe of the anchor extending end connecting band specific position mark containing specificity residue, detect the fluorescent signal of corresponding connection product, obtain the nucleotide sequence information of correspondence position;

S22. with specificity cutting agent cleavage specificity residue, by the fluorescent probe that connects in previous step and an anchor wash-out, an anchor is reset;

S23. repeat the operation of being with the connection of fluorescent probe of specific position mark and corresponding fluorescent signal to detect at an anchor extending end, obtain the sequence information of M Nucleotide after an anchor extending end.

It should be noted that, in this technical scheme, the specificity residue that described in step S21, an anchor contains and corresponding specificity cutting agent thereof can comprise multiple.

In a preferred embodiment of the invention, as shown in Figure 6, the specificity residue that an anchor contains is dU base, and the specificity cutting agent of its correspondence is UDG enzyme.

Utilize an anchor of structure as shown in Figure 6, step S2 can be realized by the method shown in Fig. 7, and the method comprises the following steps:

S21 '. at the fluorescent probe of the anchor extending end connecting band specific position mark containing dU base, detect the fluorescent signal of corresponding connection product, obtain the nucleotide sequence information of correspondence position;

S22 '. carry out enzyme cut with UDG enzyme identification dU base, by the fluorescent probe that connects in previous step and an anchor wash-out, reset an anchor;

S23 '. repeat the operation of being with the connection of fluorescent probe of specific position mark and corresponding fluorescent signal to detect at an anchor extending end, obtain the sequence information of M Nucleotide after an anchor extending end.

Step S2 is realized with this technical scheme, it is advantageous that, one anchor contains dU base, the UDG enzyme that can directly utilize specific recognition enzyme to cut dU base cuts an anchor, form short-movie section, thus to make in step S2 the wash-out of fluorescent probe and the realization of replacing easier.

In another preferred embodiment of the invention, as shown in Figure 8, an anchor with specificity residue be that the enzyme of nickase Nt.Alw I cuts identification base.

Utilize an anchor of structure as shown in Figure 8, step S2 can be realized by method as shown in Figure 9, and the method comprises the following steps:

S21 ' '. at the fluorescent probe that the anchor extending end connecting band specific position cutting recognition site containing nickase enzyme marks, detect the fluorescent signal of corresponding connection product, obtain the nucleotide sequence information of correspondence position;

S22 ' '. cut recognition site with nickase identification nickase enzyme and carry out enzyme and cut, by the fluorescent probe wash-out connected in previous step;

S23 ' '. repeat the operation of being with the connection of fluorescent probe of specific position mark and corresponding fluorescent signal to detect at an anchor extending end, obtain the sequence information of M Nucleotide after an anchor extending end.

Step S2 is realized with this technical scheme, it is advantageous that, when the fluorescent probe of belt replacement different positions mark, nickase enzyme entrained on an anchor can be utilized to cut recognition site, directly the connection between the fluorescent probe originally connected and an anchor is interrupted by corresponding nickase, thus easily the fluorescent probe originally connected is washed off, connect new fluorescent probe, avoid resetting after an anchor wash-out again, simplify the operation step, saves the cost of reagent simultaneously.

Wherein, described in above-mentioned steps, M is positive integer, and its numerical range is determined by ligase enzyme used in the present invention, is preferably 1 ~ 9, is more preferably 1 ~ 6.In one embodiment of the invention, utilize T4 ligase enzyme to realize enzyme of the present invention and cut extension sequencing, the numerical value of M is preferably 1 ~ 6; In another embodiment of the invention, utilize Tth ligase enzyme to realize enzyme of the present invention and cut extension sequencing, the numerical range of M can be preferably 1 ~ 9.In above-mentioned preferable range, the present invention utilizes enzyme to cut extension increases the sequence measurement that length is read in order-checking, reading of checking order when can not only increase detection is long, can also improve the accuracy that the fluorescent probe in sequencing procedure is connected with anchor further, and then improves the accuracy of nucleotide sequence information reading.Wherein, when utilizing T4 ligase enzyme, when fluorescent probe being connected to 3 ' end or the 5 ' end of anchor, with corresponding, 6 bases that one end connected occurs for fluorescent probe and anchor need treat that sequenced fragments complete complementary matches, could realize connecting; When utilizing Tth ligase enzyme, when fluorescent probe being connected to 3 ' end or the 5 ' end of anchor, requiring 8 of fluorescent probe and that one end that anchor connects or 9 bases respectively and corresponding treating that sequenced fragments complete complementary matches, connection could be realized.

The fluorescent probe of the band different positions mark used in above-mentioned steps is divided into different type of sets, with the different nucleotide sequence informations of the corresponding same specific position of fluorescent mark different in set type, and the specific position difference that the fluorescent probe fluorescent mark of different set type is corresponding.In each ligation, add the fluorescent probe of same set type, according to gathered fluorescent signal, the nucleotide sequence information that this group fluorescent probe mark specific position is corresponding can be obtained; And by these repetitions operated of wash-out of the connection-collection fluorescent signal-fluorescent probe of the hybridization combinations-fluorescent probe of an anchor, the sequence information of M Nucleotide after an anchor extending end can be obtained accurately.

After step S2 detects and terminates, determined nucleic acid fragment remains with an anchor of combination and the fluorescent probe of band specific position mark.Carry out enzyme for the ease of the restriction endonuclease in step S3 to cut, one anchor extending end can be reactivated, then four kinds of Nucleotide are added, utilize archaeal dna polymerase, along the direction of an anchor extending end, the determined nucleic acid fragment be fixed on solid phase carrier is formed complete double chain acid molecule.

Restriction endonuclease described in step S3, refers to and can identify that specific enzymes contained on double chain acid molecule cuts identification base sequence, then carries out the enzyme of double-strandednucleic acid cutting in the position of cutting recognition site some amount base apart from enzyme.In the present invention, can use enzyme cut after can obtain the restriction endonuclease of flat end, preferred use enzyme can obtain the restriction endonuclease of viscosity protruding terminus after cutting, its selection principle is followed by identifying that enzyme cuts recognition site, can the restriction endonuclease of all or part of excision by the nucleotide sequence obtained in step S2.When selecting restriction endonuclease, preferred optimal reactive temperature is at the restriction endonuclease of about 37 DEG C, and the too high restriction endonuclease of optimal reactive temperature easily causes the sex change cutting through journey double center chain at enzyme to be dissociated, thus causes the connection of follow-up fluorescent probe to be obstructed; Preferably to insensitive restriction endonuclease that methylates, so that the nucleotide sequence of determined nucleic acid fragment is cut and obtained to the enzyme that can continue; Preferred enzyme cuts Nucleotide number between recognition site and restriction enzyme site more than 4, make each enzyme cut after to extend forward the length of order-checking longer.

The present invention's restriction endonuclease used can be II type restriction endonuclease, include but not limited to Acu I, Alw I, Bbs I, Bbv I, Bcc I, BceA I, BciV I, BfuA I, Bmr I, Bpm I, Bsa I, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspM I, BspQ I, BtgZ I, Ear I, Eci I, Fau I, Fok I, Hga I, Hph I, HpyA V, Mbo II, Mly I, Mnl I, Ple I, Sap I, SfaN I, BpuE I, Mme I and NmeA III, wherein preferred Acu I, Bbv I, BceA I, Bpm I, BseR I, BspM I, Fok I, Hga I, Mbo II and Mnl I, restriction endonuclease used also can be III type restriction endonuclease, includes but not limited to Ecop1 and Ecop15 I.

In step S3, cut recognition site for the enzyme being undertaken identifying by restriction endonuclease, can be brought into by an anchor, also can be brought into by additive method.Cut the difference in recognition site source according to enzyme, step S3 also can be realized by diverse ways.

In an embodiment in the present invention, enzyme is cut recognition site and is directly brought into by an anchor, and namely an anchor contains at least one enzyme and cuts recognition site.

Utilize enzyme contained on an anchor to cut recognition site, multiple different mode can be had to realize step S3.In an embodiment of this programme, step S3 can directly utilize on restriction endonuclease identification the one anchor with enzyme cut recognition site, by having obtained the nucleotide segment of sequence information in step S2 or having excised completely, obtain the digestion products containing treating sequenced fragments.

The advantage utilizing this embodiment to realize step S3 is, after step S2 obtains an anchor extending end M Nucleotide sequence information after, to utilize on an anchor contained enzyme to cut recognition site and directly carry out enzyme and cut, can simplify the operation step.

In another embodiment of this programme, utilize enzyme contained on an anchor to cut recognition site, step S3 can also be realized by method as shown in Figure 10, and the method comprises the steps:

S31. by the fluorescent probe that connects in step S2 and an anchor wash-out, reset an anchor and also carry out chain extension, form double chain acid molecule with determined nucleic acid fragment;

S32. restriction endonuclease by identification the one anchor with enzyme cut recognition site, by having obtained the nucleotide segment of sequence information in step S2 or having excised completely, obtained the digestion products containing treating sequenced fragments.

The advantage utilizing this technical scheme to realize step S3 is, first utilizes the extending end of an anchor to carry out chain extension, extends to form double chain acid molecule with determined nucleic acid fragment, can ensure that digestion products keeps double-stranded state, be convenient to the connection of follow-up joint.

In a preferred embodiment of the present embodiment, determined nucleic acid fragment is connected and fixed on microballon by 5 ' end, and the anchor being incorporated into the first joint cuts recognition site sequence 5 ' containing enzyme ... CTGAAG ... 3 ', utilizing II type restriction endonuclease Acu I to identify in step S3, this enzyme is cut recognition site and is carried out enzyme cuts, and obtains treating that sequenced fragments 3 ' holds the digestion products containing two outstanding Nucleotide.

In another preferred embodiment of the present embodiment, determined nucleic acid fragment is connected and fixed on microballon by 5 ' end, and the anchor being incorporated into the first joint cuts recognition site sequence 5 ' containing enzyme ... GCAGC ... 3 ', utilizing II type restriction endonuclease Bbv I to identify in step S3, this enzyme is cut recognition site and is carried out enzyme cuts, and obtains treating that sequenced fragments 3 ' holds the digestion products containing two outstanding Nucleotide.

In another preferred embodiment of the present embodiment, determined nucleic acid fragment is connected and fixed on microballon by 5 ' end, and the anchor being incorporated into the first joint cuts recognition site sequence 5 ' containing enzyme ... GAGTC ... 3 ', utilizing II type restriction endonuclease Mly I to identify in step S3, this enzyme is cut recognition site and is carried out enzyme cuts, and obtains treating that sequenced fragments 3 ' holds the digestion products containing flat end.

In a preferred embodiment of the present embodiment, determined nucleic acid fragment is connected and fixed on microballon by 3 ' end, and the anchor being incorporated into the first joint cuts recognition site sequence 5 ' containing enzyme ... CATCC ... 3 ', utilize this enzyme of Fok I enzyme identification to cut recognition site and carry out enzyme in step S3 to cut, obtain treating that sequenced fragments 5 ' holds the digestion products containing 4 outstanding Nucleotide.

In a specific embodiment of the present embodiment, determined nucleic acid fragment is connected and fixed on microballon by 5 ' end, and the anchor being incorporated into the first joint cuts recognition site sequence 5 ' containing enzyme ... CAGCAG ... 3 ', utilize III this enzyme of type restriction endonuclease Ecop15 I enzyme identification to cut recognition site and carry out enzyme in step S3 to cut, obtain treating that sequenced fragments 3 ' holds the digestion products containing two outstanding Nucleotide.

In another embodiment of the invention, enzyme is cut recognition site and is brought into by the 3rd joint cutting recognition site containing at least one enzyme in the other end connection of an anchor.Utilize the 3rd joint, step S3 can be realized by method as shown in figure 11, and the method comprises the following steps:

S31 '. connect the 3rd joint of double-strand at the other end of an anchor, the 3rd joint contains at least one enzyme and cuts recognition site;

S32 '. utilize restriction endonuclease identification the 3rd joint with enzyme cut recognition site, by having obtained the nucleotide segment of sequence information in step S2 or having excised completely, obtained the digestion products containing treating sequenced fragments.

It should be noted that, the other end of a described anchor, be in relative step S1 an anchor for the end that extends; If the other end of an anchor was closed before step S3, so needed first to activate before step S3; Activation method can utilize of the prior art any one, only the group that can be used in the other end connecting need be come out, as in a preferred embodiment of the present invention, one anchor itself, containing specificity residue, can directly utilize specificity cutting agent cleavage specificity residue to complete activation.Described 3rd joint, is the nucleic acid molecule cutting recognition site containing at least one enzyme, cuts recognition site, so that in subsequent step, will be obtained the nucleotide segment of sequence information by restriction endonuclease or excise completely for the enzyme introducing restriction endonuclease.The advantage utilizing this technical scheme to realize step S3 for suitability wide, to nucleic acid fragment to be measured without particular requirement, whether the anchor no matter determined nucleic acid fragment combined cuts recognition site containing enzyme, all realizes by this technical scheme.

What needs further illustrated is, 3rd joint can select the corresponding joint containing different end, specifically can include but not limited to the joint of flat end fitting, protruding terminus joint, branch type joint and band loop-stem structure, the structure of these joints and characteristic can see patent documentations: CN201110222952.4.Preferably use the joint of protruding terminus joint, branch type joint and band loop-stem structure in the present invention, the joint of these structures can be avoided occurring between multiple joint from the phenomenon connected in connection procedure.For above-mentioned different implementation, on 3rd joint, the described endonuclease digestion recognition site cut for enzyme can be any one corresponding to above-mentioned restriction endonuclease, the Nucleotide only need cut between recognition site and restriction enzyme site by enzyme when design and synthesis is precalculated, make enzyme cut after just will read the nucleotide segment of sequence information in step S2 or excise completely, the excision different enzymes therefore can being selected in the present invention to cut recognition site and corresponding restriction endonuclease thereof realize the Nucleotide having obtained sequence information in step S2.

Should be noted that; above-mentioned embodiment is only cut some specific embodiments of recognition site and corresponding restriction endonuclease thereof in the present invention about the enzyme selected; not in order to limit the scope of the invention; use other enzymes qualified instead and cut recognition site and corresponding restriction endonuclease, object of the present invention can be realized equally.

For obtaining the digestion products containing treating sequenced fragments, purifying recovery can be carried out after the endonuclease reaction of step S3, and the method that purifying reclaims can utilize various ways of the prior art to realize.In one embodiment of the invention, determined nucleic acid fragment is fixed on slide, and enzyme directly can realize needing the digestion products of sequenced fragments and other separating substances purifying with wash buffer after cutting; In another specific embodiment of the present invention, determined nucleic acid fragment is fixed on magnetic bead, directly utilizes magnet adsorption, slightly rinses with damping fluid, can realize digestion products and other separating substances purifying containing treating sequenced fragments.

In rapid S4, before connection second joint, according to the difference of the digestion products obtained in step S3 and the method for attachment of follow-up use, optionally can carry out moditied processing, as end-filling to digestion products; Also can directly utilize enzyme cut after the protruding terminus that obtains carry out the connection of the second joint.

In one particular embodiment of the present invention, according to utilize in step S3 on an anchor the enzyme with restriction endonuclease cut recognition site, carry out enzyme with Fok I and cut, obtain the digestion products of 3 ' end containing 4 nucleotide sequence protruding terminuses.First end-filling process is carried out to digestion products, and then connects the second joint.The advantage of this embodiment is can avoid making the kind of the second joint synthesis too much due to long protruding terminus, thus reduces the synthesis cost of the second joint.

In another specific embodiment of the present invention, according to utilize in step S3 on an anchor cut recognition site for the enzyme of Acu I with enzyme and cut recognition site, carry out enzyme with Acu I and cut, obtain 3 ' digestion products containing 2 nucleotide sequence protruding terminuses.The Nucleotide outstanding for this protruding terminus is less, only need synthesize 4 ²kind of the second joint can realize connecting, therefore directly utilize enzyme cut after the protruding terminus that obtains carry out the connection of the second joint.The advantage of this embodiment is the step that can simplify the operation, and directly adds the second joint and carries out reacting.

In step S4, described second joint, be for containing treating that the digestion products of sequenced fragments is connected to form the double chain acid molecule of new determined nucleic acid fragment.According to digestion products end and the mode of connection selected different, second joint can select the corresponding joint containing different end, include but not limited to the joint of flat end fitting, protruding terminus joint, branch type joint and band loop-stem structure, the structure of these joints and characteristic can see patent documentations: CN201110222952.4.Preferably use the joint of protruding terminus joint, branch type joint and band loop-stem structure in the present invention, the joint of these structures can be avoided occurring between multiple joint from the phenomenon connected in connection procedure.

In step S4, the complementary pairing for the ease of follow-up 2nd anchor and the second joint can carry out smoothly, and the double-strandednucleic acid formed after can being connected with the second joint digestion products connects product and processes, and forms the new determined nucleic acid fragment of single stranded form.Double-strandednucleic acid to be connected on product complementary be incorporated into treat Nucleotide on sequenced fragments and enzyme cut after the Nucleotide that remains carry out the method that process forms the new determined nucleic acid fragment of strand, include but not limited to be removed by NaOH sex change dissociate or the heat up mode of annealing.

Described 2nd anchor is the single stranded nucleic acid molecule that can be combined with the second joint grappling, for connecting fluorescent probe in step s 5; Described 2nd anchor can be identical or different from an anchor.If the 2nd anchor is identical with an anchor, identical restriction endonuclease can be used to operate when then follow-up continuation enzyme is cut, simplify the kind of reaction reagent, can also avoid, because there is identical endonuclease sequence in new determined nucleic acid fragment, causing follow-up enzyme to cut step and obtaining non-targeted product simultaneously; If the 2nd anchor is different from an anchor, then can introduces new design in the synthesis of the 2nd anchor, meet more actual needs.2nd anchor is different from an anchor's, and it is different with kind to be that enzyme cuts the position of recognition site, also can be the difference of Nucleotide quantity.

2nd anchor can carry out other process as required.In one particular embodiment of the present invention, as required 3 ' of the 2nd anchor end or 5 ' end are closed, in order to control linkage direction, and avoid the 2nd anchor certainly connecting each other.In an embodiment of the present embodiment, hold the method closed to include but not limited to two deoxidation, ammoxidation, amidation to 3 ', it cannot continue to connect, and the connection controlling the 2nd anchor only occurs in 5 ' end; In another embodiment of the present embodiment, 5 ' of the 2nd anchor end is closed, carry out dephosphorylation or amidation process is closed by 5 ' end, thus only retain the end of 3 ' end as connection.

In another specific embodiment of the present invention, the 2nd anchor contains specificity residue, can make the replacing of follow-up fluorescent probe and wash-out easier.In a preferred embodiment of the present invention, 2nd anchor with specificity residue be dU base, several dU base is introduced in its nucleotide sequence, 2nd anchor of this structure can directly excise dU base and form different short-movie sections in the elution process of follow-up order-checking, is convenient to the realization of wash-out.

In another preferred embodiment of the present invention, 2nd anchor with specificity residue be base containing phosphorothioate bond, in its nucleotide sequence, replace original P-O key with one or more phosphorothioate bond (P-S), can directly utilize the cutting of the compound containing Ag, Hg, Cu, Mn, Zn and Cd ion P-S key to realize the wash-out of the 2nd anchor.

In another preferred embodiment of the present invention, 2nd anchor contains two enzymes and cuts recognition site, one of them is digestion with restriction enzyme recognition site for cutting double-strand, and another be that nickase enzyme for cutting a strand formation otch in double-strand cuts recognition site.

It should be noted that above-described embodiment is only some embodiments of the 2nd anchor in the present invention, not in order to limit the scope of the invention.

In step S5, after obtaining the 2nd anchor extending end, the sequence information of N number of Nucleotide is realized by method as shown in figure 12, and the method comprises the following steps:

S51. the fluorescent probe that connecting band specific position marks after the 2nd anchor extending end, detects the fluorescent signal of corresponding connection product, obtains the nucleotide sequence information of corresponding position;

S52. removed by the fluorescent probe that previous step connects, the fluorescent probe of connecting band different positions mark, detects the fluorescent signal of corresponding connection product, obtains the nucleotide sequence information of respective markers position;

S53. the operation of repeating step S52, until the sequence information of N number of Nucleotide after obtaining the 2nd anchor extending end.

Connection between the fluorescent probe that technique scheme utilizes band different positions to mark and replacing, realize the acquisition to the sequence information of N number of Nucleotide after the 2nd anchor extending end.Wherein, the removal of fluorescent probe described in step S52 can have various ways to realize.

In an embodiment of this step, NaOH sex change is utilized fluorescent probe and the 2nd anchor to be disintegrated down from new determined nucleic acid fragment, then by the 2nd anchor recombine on the second joint, carry out the connection of the different fluorescent probe in succeeding marker position and signal detection operation.This embodiment is simple, does not need to add other reagent.

In another preferred implementation of this step, 2nd anchor contains several dU base, therefore UDG enzyme is utilized directly to carry out the excision of dU base, form short-movie section, intensification sex change is dissociated, again obtain new determined nucleic acid fragment, then by the 2nd anchor recombine on the second joint, carry out the connection of the different fluorescent probe in succeeding marker position and signal detection operation.

In another preferred implementation of this step, in the nucleotide sequence of the 2nd anchor, several thiophosphoric acid key (P-S) replaces original P-O key, therefore directly utilizes the cutting of the compound containing Ag, Hg, Cu, Mn, Zn and Cd ion P-S key to realize the removal of the 2nd anchor and fluorescent probe.

In another preferred implementation of this step, the enzyme that a nickase is contained at 2nd anchor extending end place cuts recognition site, therefore nickase is directly utilized the connection between fluorescent probe and the 2nd anchor to be removed, just can continue the different fluorescent probe in linkage flag position at the extending end of the 2nd anchor, realize the enforcement of follow-up sequencing steps.Fluorescent probe is directly removed by this embodiment, and does not need the 2nd anchor process, has both simplified the operation, has reduced reagent cost again.

Above-mentioned preferred implementation utilizes specific material by the connection excision in the 2nd anchor, forms short and small fragment, then utilizes mild conditions to remove fluorescent probe.

Wherein, N described in above-mentioned steps is positive integer, and its numerical range is determined by ligase enzyme used in the present invention equally, is preferably 1 ~ 9, is more preferably 1 ~ 6.In one embodiment of the invention, utilize T4 ligase enzyme to realize enzyme of the present invention and cut extension sequencing, the numerical value of N is preferably 1 ~ 6; In another embodiment of the invention, utilize Tth ligase enzyme to realize enzyme of the present invention and cut extension sequencing, the numerical range of N is preferably 1 ~ 9.In above-mentioned preferable range, enzyme of the present invention is cut and is extended sequencing and can not only increase order-checking and read long, can also improve the accuracy that the fluorescent probe in sequencing procedure is connected with anchor further, and then the accuracy of raising nucleotide sequence information reading.Wherein, when utilizing T4 ligase enzyme, when fluorescent probe being connected to 3 ' end or the 5 ' end of anchor, with corresponding, 6 bases that one end connected occurs for fluorescent probe and anchor need treat that sequenced fragments complete complementary matches, could realize connecting; When utilizing Tth ligase enzyme, when fluorescent probe being connected to 3 ' end or the 5 ' end of anchor, requiring 8 of fluorescent probe and that one end that anchor connects or 9 bases respectively and corresponding treating that sequenced fragments complete complementary matches, connection could be realized.

Figure 13 is the method schematic diagram of the sequence information obtaining N number of Nucleotide after the 2nd anchor extending end in step S5 in a specific embodiment, and this figure presents the process of the sequence information obtaining N number of Nucleotide after the 2nd anchor extending end intuitively.

Step 51. reads the 1st nucleotide sequence information after the 2nd anchor extending end: under the effect of T4 ligase enzyme, after 2nd anchor extending end, linkage flag position is the fluorescent probe of the 1st, then figure is adopted, collect fluorescent signal, determine the nucleotide sequence information of after the 2nd anchor extending end the 1st according to the fluorescent signal obtained.

2nd nucleotide sequence information after step 52. reads the 2nd anchor extending end: utilize the 2nd anchor extending end place with nickase enzyme cut recognition site, connection between fluorescent probe and the 2nd anchor is cut away, being dissociated mark position by intensification sex change is the fluorescent probe wash-out of the 1st, then using mark position instead is the extending end that the fluorescent probe of the 2nd is connected to the 2nd anchor, adopt figure imaging, collect fluorescent signal, determine the nucleotide sequence information of after the 2nd anchor extending end the 2nd according to the fluorescent signal obtained.

Step 53. reads succeeding nucleotide: the operation of repeating step 52, and the fluorescent probe using band different positions mark instead obtains the nucleotide sequence information of corresponding position, until the sequence information of the 6th Nucleotide after obtaining the 2nd anchor extending end.

In above-mentioned embodiment, the nickase enzyme utilizing the 2nd anchor extending end place to contain cuts recognition site, the connection directly realizing the fluorescent probe of band different positions mark is changed, thus the sequence information of 6 Nucleotide after realizing acquisition the 2nd anchor extending end.

It should be noted that above-described embodiment is only some specific embodiments of the sequence information realizing obtaining in step S5 N number of Nucleotide after the 2nd anchor extending end, not in order to limit the scope of the invention.Such as change the numerical value of wherein N, as change from react before in the different reagent of the reagent that uses or reagent reacted in reacting before is changed into the reagent that unreacted crosses.

The reagent changed described in step S6, refers to the fluorescent probe of restriction endonuclease, joint, anchor and band different positions mark used in step S3 to S5; Described replacing, refers to the replacing of broad sense, as changed different types of reagent or reacted reagent being changed into the reagent that unreacted crosses.

After reagent is changed, with restriction endonuclease, the product of step S5 is read to the excision of nucleotide sequence, and then digestion products is connected with new joint, and in conjunction with new anchor, connect new fluorescent probe, detect the fluorescent signal of this fluorescent probe, the nucleotide sequence of this fluorescent probe correspondence position can be obtained.So carry out similar above-mentioned cyclical operation, nucleotide sequence information required in determined nucleic acid fragment can be obtained.

Figure 14 increases order-checking to read long sequence measurement schematic diagram in one embodiment of the invention, this figure presents the process that the method utilizing enzyme to cut extension carries out checking order intuitively.

Step 1. the one anchor hybridizes combination: be incorporated on the first joint of determined nucleic acid fragment by an anchor by base pair complementarity hybridization, and wherein 5 ' end of determined nucleic acid fragment is by being connected and fixed on bead surface; Wherein, an anchor contains sequence is 5 ' ... CTGAAG ... the enzyme of 3 ' cuts recognition site, and containing dU base in the nucleotide sequence of an anchor.

Step 2. obtains the sequence information of 6 Nucleotide after an anchor extending end: under the effect of T4 ligase enzyme, the fluorescent probe for detecting is connected at the extending end of an anchor, detect the nucleotide sequence information that fluorescent signal obtains corresponding position, and utilize UDG enzyme to excise dU base with the replacing detection of the fluorescent probe of the replacement and band different positions mark that realize an anchor, gather the fluorescent signal figure of correspondent probe, obtain the nucleotide sequence information of the 1 to 6.

Step 3. enzyme is cut: the carrying out cut for ease of follow-up enzyme, after the sequence information of 6 Nucleotide is all read after an anchor extending end, first under the effect of archaeal dna polymerase, determined nucleic acid fragment is extended to form complete double chain acid molecule; Then Acu I enzyme spcificity identification is utilized and enzyme to be cut in an anchor entrained enzyme and cut recognition site, 6 nucleotide sequences of the anchor extending end read before are excised, obtains the non-sequenced fragments of 3 ' end containing two Nucleotide protruding terminuses; After endonuclease reaction, utilize magnet adsorption magnetic bead, the digestion products purifying containing non-sequenced fragments is reclaimed.

Step 4. digestion products connects the second joint and in conjunction with the 2nd anchor: two Nucleotide protruding terminuses contained by the one end utilizing the digestion products of step 3, and is altogether 4 ²second joint of=16 kinds, carries out connection and obtains double-strand connection product under the effect of T4 ligase enzyme; After ligation terminates, double-strand is connected product sex change and to dissociate formation strand, be fixed the new determined nucleic acid fragment on magnetic bead; 2nd anchor is incorporated on the second joint of new determined nucleic acid fragment by base pair complementarity.Wherein, same on the 2nd anchor is 5 ' containing sequence ... CTGAAG ... the enzyme of 3 ' cuts recognition site, and distance the 2nd nucleotide position place of 4, anchor extending end place also containing sequence be 5 ' ... GGATC ... the enzyme of 3 ' cuts recognition site.

Step 5. obtains the sequence information of 6 Nucleotide after the 2nd anchor extending end: according to the method for embodiment as shown in figure 12, connection between the fluorescent probe utilizing nickase Nt.Alw I cutting belt specific position to mark and the 2nd anchor, realize the replacing of band different positions mark fluorescent probe, thus the nucleotide sequence information of different positions can be read, the sequence information of 6 Nucleotide after acquisition the 2nd anchor extending end.

Step 6. changes reagent, operation in repeating step 3, step 4, step 5, after obtaining the nucleotide sequence information of some amount (its numerical range is preferably 1 ~ 6), the nucleotide sequence read is excised by enzyme means of cutting, and build new determined nucleic acid fragment, the object extending order-checking is realized, until obtain nucleotide sequence information required in determined nucleic acid fragment with this.

For above-mentioned each technical scheme, for further illustrating technique effect and the superiority of technical scheme described in the present invention, the present invention provides a concrete operation embodiment.

The present embodiment includes milk with one and absorbs the mono-clonal plasmid with one section of nucleic acid fragment (SEQ ID NO:103) of digestion ability MCM6 gene, and nucleic acid-templated as testing sample of mono-clonal plasmid including one section of nucleic acid fragment (SEQ ID NO:104) of folic acid and vitamins B receptivity mthfr gene.Wherein, MCM6 gene and the corresponding specificity amplification primer of mthfr gene are: SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, therefrom amplify the target nucleic acid fragment for follow-up order-checking respectively, then utilize target nucleic acid fragment to build sequencing library, thus carry out the detection of determined nucleic acid fragment.

Wherein 5 ' end connector of determined nucleic acid fragment is combined with magnetic bead by the effect of the affine vitamin H of strepto-, and 3 ' end of determined nucleic acid fragment is containing the first joint sequence.For reducing the complicacy of operating process, the first joint of two kinds of nucleic acid fragments adopts identical sequence, and the anchor that therefore both use in the present embodiment is also the same, and both operate simultaneously.The Pstar-II Plus order-checking platform that the Treatment Analysis of whole sequencing procedure and fluorescent signal view data adopts Shenzhen HYK Gene Technology Co., Ltd. to produce carries out.Described embodiment concrete operations are as follows.

One, the amplification of determined nucleic acid fragment.

Utilizing above-mentioned nucleic acid-templated specificity amplification primer, the target nucleic acid fragment of MCM6 gene and mthfr gene is increased, obtaining the target nucleic acid fragment for building sequencing library.Wherein, described amplification is carried out respectively, and reaction system is as follows: F primer (10 μMs), 2 μ L; R swims primer (10 μMs), 2 μ L; The each 2.5mM of dNTP(), 4 μ L; As nucleic acid-templated plasmid, 20ng; Ex Taq(5U/ μ L), 0.25 μ L; 10 × Ex Taq Buffer, 5 μ L; ddH ₂o adds to 50 μ L.

PCR reaction conditions is as follows:

95℃ 3min；

94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s; Repeat 25 circulations;

72℃ 7min。

Utilize PCR to reclaim test kit, be separated respectively to the amplified production of each sample, remove the primer and dNTP that do not increase, agarose gel purification reclaims target nucleic acid fragment.

Two, the target nucleic acid fragment obtained that increases is utilized to build sequencing library.

Target nucleic acid product two ends obtained of increasing in step one connect the first joint, then carry out unit molecule amplification, obtain the sequencing library checked order.

1. target nucleic acid fragment is connected with the first joint.

The protruding terminus joint of outstanding T base is contained as the first joint using end, its concrete sequence is SEQ ID NO:95 and SEQ ID NO:96, and the biotinylation that this first joint contains specific position is modified, so that the follow-up magnetic bead with modifying containing Streptavidin is connected.First joint is connected with target nucleic acid fragment, obtains the target nucleic acid fragment containing the first joint.

Under the effect of T4 ligase enzyme, the two kinds of target nucleic acid products obtained increasing respectively mix with equimolar ratio, are connected with the first joint, obtain the target nucleic acid fragment containing the first joint, linked system is: the mixture of target nucleic acid product, and 50 μ L(are about 500ng); First joint, 2 μ L(are about 3000ng); 10mM ATP, 5 μ L; T4 DNA ligase (30U/ μ L), 1 μ L; 10 × T4 ligase enzyme damping fluid, 10 μ L; Add ddH ₂o to 100 μ L.

Hatch more than 4h for 16 DEG C, after reaction terminates, utilize Purification Kit to reclaim.

2. utilize the target nucleic acid fragment containing the first joint to carry out unit molecule amplification, build sequencing library.

Utilize the amplification method as shown in Fig. 3 before, carry out unit molecule amplification with the target nucleic acid fragment containing the first joint, build sequencing library, obtain determined nucleic acid fragment, concrete operations are as follows.

1) by the target nucleic acid fragment containing biotin modification and the Myone magnetic bead modified containing Streptavidin (1 μm, 10mg/mL; Invitrogen) combine, make magnetic bead surfaces be fixed with at least one target nucleic acid fragment, reaction system and reaction process are: the target nucleic acid fragment containing the first joint, 0.018ng(10 ⁸individual molecule); Myone magnetic bead (1 μm, 10mg/mL; Invitrogen), 6 μ L; Spiral vibration mixing, reaction 30min, with appropriate TE damping fluid (10mM Tris-HCl, pH8.0; 1mM EDTA) clean twice, centrifugation, by the magnetic bead that obtains with 6 μ L binding buffer liquid (10mM Tris-HCl, pH7.5; 1mM EDTA; 1M NaCl; 0.01% Triton X-100) resuspended preservation.

2) unit molecule amplimer is incorporated into magnetic bead surfaces.

Product step 1) obtained and 5 ' is held to contain biotin labeling and hold the 6th Nucleotide to contain amidized unit molecule amplimer (F3, R3) apart from 5 ' and is reacted, make biotinylated F3, R3 and the target nucleic acid fragment containing the first joint be combined in magnetic bead surfaces simultaneously, wherein, the sequence of F3, R3 is SEQ ID NO:97 and SEQ ID NO:98, complementary with the target nucleic acid fragment containing the first joint.Reaction system and process as follows: biotinylation and amidized primers F 3(100 μM), 0.3 μ L; Biotinylation and amidized primer R3(100 μM), 0.3 μ L; The bead suspension obtained in step 1), 6 μ L; Under room temperature condition (18 ~ 25 DEG C), spiral vibrates, and hatches 1h; Appropriate TE buffer solution for cleaning 2 times, centrifugal, with the resuspended magnetic bead of 6 μ L TE damping fluid, obtain bead suspension, 4 DEG C save backup.

In this step, for being combined with magnetic bead, and roughly the same with the quantity of magnetic bead as the mole number of the target nucleic acid fragment of unit molecule amplification template sequence, therefore, a magnetic bead surfaces in the bead suspension of step 1) gained only combines minute quantity or even single template sequence.

3) for the preparation of the emulsion system of unit molecule amplification.

Adopt the KE001 emulsion of Shenzhen HYK Gene Technology Co., Ltd. to prepare test kit, prepare emulsion system according to operation instruction.First prepare reagent to oil phase to mix with spiral thermal agitation, be placed in room temperature 30min, obtain the oil-based system for the preparation of emulsion system; Utilizing step 2) bead suspension that obtains prepares PCR Mix aqueous phase system, and for 150 μ L, this aqueous phase system is as follows: ddH ₂o, 113 μ L; 10 × PCR buffer(650mM Tris-HCl, pH8.0; 160mM (NH ₄) ₂sO ₄; 10mM DTT; 11mM MgCl ₂), 15 μ L; 50mM MgSO ₄, 3 μ L; 10mM dNTP, 3 μ L; Non-biotinylation and without amidized F3(10 μM), 0.5 μ L; Non-biotinylation and without amidized R3(10 μM), 0.5 μ L; Step 2) bead suspension that obtains, 6 μ L; 5U/ μ L DNA Taq enzyme, 9 μ L; Mentioned component is mixed, is prepared into PCR Mix aqueous phase system.

The oil-based system prepared is put into EP pipe with aqueous phase system according to the ratio of 4:1 mix, add the steel ball of auxiliary mixing simultaneously, EP pipe is placed on emulsion preparing instrument and clamps, according to 15HZ, 10s, change 17HZ again, 8s carries out vibration mixing, is prepared into the emulsion system for unit molecule amplification.

4) emulsion system prepared is utilized to carry out unit molecule amplification.

Utilize the emulsion system for preparing to carry out the amplification of EPCR unit molecule, reaction system and reaction process as follows:

4min，94℃；

30s，94℃，

55s，64℃，

45s, 72 DEG C, cycle number is 3;

30s，94℃，

55s，61℃，

45s, 72 DEG C, cycle number is 3;

30s，94℃，

55s，58℃，

45s, 72 DEG C, cycle number is 3;

30s，94℃，

55s，57℃，

45s, 72 DEG C, cycle number is 100;

6min，72℃；

Reaction terminates rear 10 DEG C of preservations.

3. breakdown of emulsion release amplified production, separating-purifying obtains sequencing library.

Add appropriate Virahol in reaction product after EPCR reaction terminates, after spiral concussion mixing, supernatant is removed in 4000rpm, 3min centrifugation, and amplified production is with magnet adsorption.

Add appropriate extraction buffer (Extraction buffer) in amplified production, the centrifugal 3min layering of 4000rpm after spiral vibration mixing, uses magnet adsorption magnetic bead, by alveolar fluid clearance; Repeat this operation for several times.

Then add appropriate TE, repeated washing number, all over amplified production, finally with the resuspended magnetic bead of appropriate TE, obtains the magnetic bead containing determined nucleic acid fragment.

Three, utilize sequencing library to carry out enzyme and cut extension order-checking.

1. an anchor grappling is incorporated on the first joint of determined nucleic acid fragment.

Magnetic bead containing determined nucleic acid fragment is mixed with order-checking damping fluid, the slide modified carries out point sample fix containing carboxy activating group, form order-checking array.By an anchor(SEQ ID NO:99) and the first joint of determined nucleic acid fragment between to be hybridized by base pair complementarity and combine, enzyme wherein containing Acu I enzyme on an anchor extending end cuts recognition site, this reaction process and system are: 28 DEG C, 400 μ L 2 × SSPE(saline sodium phosphate EDTA) [175.32g/L NaCl, 31.202g/L NaH ₂pO ₄.2H ₂o, 0.01M EDTA, pH7.4] hybridization buffer carries out rinse to the determined nucleic acid fragment being fixed on surface of glass slide; Add an anchor(15 μM), be warming up to 65 DEG C under 2 × SSPE environment, maintain 30s; Be cooled to 42 DEG C, hybridization 1min; With magnet adsorption magnetic bead, 30 DEG C, 900 μ L cleaning buffer solutions [50mM KCl, 10mM Tris-HCl(pH7.4), 0.1mM EDTA] clean, a unreacted anchor is separated and removes.

2. obtain the sequence information of 6 Nucleotide after an anchor extending end.

1) fluorescent probe is connected.

The present embodiment fluorescent probe used is divided into different type of sets, with the different IPs nucleotide sequence of the corresponding same specific position of fluorescent mark different in set type, and the specific position difference that the fluorescent probe fluorescent mark of different set type is corresponding.In each ligation, add the fluorescent probe of same set type, according to gathered fluorescent signal, the nucleotide sequence information that this group fluorescent probe mark specific position is corresponding can be read.The structure of described fluorescent probe is 5 '-NNNXNNNNN-3 ', and wherein N is degeneracy base, and X is any one in A, T, G and C, X can 5 ' terminal number rise the 1st to the 6th in any position.

Under the effect of T4 ligase enzyme, after being connected to an anchor extending end after different for correspondence No. 1 bit strip fluorescently-labeled one group of four kinds of fluorescent probe mixing, ligation process and system are: 30 DEG C, connect damping fluid [100mM MgCl ₂.6H ₂o, 10mM Tris-HCl(pH7.4)] rinse is carried out to the product that hybridization is separated; Add 0.2U/ μ L T4 ligase enzyme, fluorescent probe (concentration is 2.5 μMs), connect in damping fluid, 30 DEG C, reaction 20min; After ligation terminates, with magnet adsorption magnetic bead, 30 DEG C, 900 μ L cleaning buffer solutions clean along slide plane, are separated by unreacted fluorescent probe with connection product.

2) gather fluorescent signal, read the nucleotide sequence information of corresponding position.

The connection product being connected with fluorescent probe is put into sequenator, excites under fluorescent microscope, gather fluorescent signal, judge according to fluorescent signal the nucleotide sequence reading this position.

3) wash-out the one anchor and fluorescent probe.

After the nucleotide sequence information reading corresponding position in previous step, diverse ways can be utilized to carry out wash-out.

In the present embodiment, utilize the direct sex change of NaOH to dissociate, reaction process and system are: add NaOH(0.05M in sequencing reaction system), sex change 30s; With magnet adsorption magnetic bead, 30 DEG C, 900 μ L elution buffers clean, and anchor sex change dissociated and fluorescent probe and NaOH carry out cleaning and be separated.

In another embodiment of the present invention, utilize UDG enzyme to carry out cutting to the dU base in an anchor and form small segment, then by direct for small segment wash-out, reaction process and system are: enzyme cutting buffering liquid, 20 μ L; UDG enzyme, 10 μ L; Elution buffer adds to 200 μ L; 37 DEG C, 5min carries out enzyme and cuts; After enzyme cuts end, with magnet adsorption magnetic bead, add 900 μ L elution buffers and carry out wash-out separation, do not connect the determined nucleic acid fragment of an anchor.

4) repeat above-mentioned steps, connected by the fluorescent probe changing different mark position, thus read the nucleotide sequence information obtaining after an anchor extending end the 1 to 6.

3.Acu I enzyme is cut, and obtains the digestion products containing non-sequenced fragments.

1) double chain acid molecule is extended to form.

For ease of the carrying out that follow-up enzyme is cut, after after an anchor extending end, 6 nucleotide sequence informations are all read, NaOH sex change dissociating method removing fluorescent probe described before utilization and an anchor, and recombine the one anchor is on the first joint, then under the effect of archaeal dna polymerase, determined nucleic acid fragment is extended to form complete double chain acid molecule, extension system and process are: under 30 DEG C of conditions, with the binding substances of Klenow enzyme reaction buffer solution rinse the one anchor and determined nucleic acid fragment; Add the Klenow enzyme (BioLabs of dNTP and 0.1U/ μ L inc., article No. M0210L), in 1 × NEBuffer2 reaction buffer, 37 DEG C, hatch 10min; After extension terminates, with magnet adsorption magnetic bead, 30 DEG C, 900 μ L cleaning buffer solutions clean, and are separated the complete double chain acid molecule obtaining determined nucleic acid fragment and extend to form.

2) Acu I enzyme is cut.

After obtaining double chain acid molecule, carrying out enzyme and cut, obtaining the digestion products containing treating sequenced fragments with Acu I, the reaction system that enzyme is cut and process are: 30 DEG C, add 400 μ L 1 × NEBuffer, 2,40 μMs of SAM in double chain acid molecule; Add the Acu I enzyme (BioLabs of 0.05U/ μ L inc.), 2h is hatched for 37 DEG C; After endonuclease reaction terminates, with magnet adsorption magnetic bead, 30 DEG C, 900 μ L cleaning buffer solutions clean, and are separated the digestion products obtained containing treating sequenced fragments, wherein containing treating that the nucleotide chain 3 ' of sequenced fragments holds the protruding terminus containing two Nucleotide.

4. digestion products connects the second joint, and in conjunction with the 2nd anchor.

1) digestion products connects the second joint.

Utilize digestion products with protruding terminus, be connected with second joint (SEQ ID NO:100 and SEQ ID NO:101) of structure as shown in figure 14, wherein-X the base of protruding terminus is A or G or C or T, and the second joint is altogether for 4 with kind ²the form of=16 kinds of joint equimolar ratio mixing adds in ligation, and the system of ligation and process are: under 30 DEG C of conditions, adds 1 × connection damping fluid in the digestion products reclaimed; Add the second joint that concentration is 16 kinds of joint mixing of 10 μMs, and the T4 ligase enzyme of 0.2U/ μ L, hatch 1h under 16 DEG C of conditions; After ligation terminates, with magnet adsorption magnetic bead, 30 DEG C, 900 μ L cleaning buffer solutions clean, and are separated the new determined nucleic acid fragment obtaining double chain acid molecule form.

2) combination of the 2nd anchor.

With the method that NaOH sex change is dissociated, the new determined nucleic acid fragment of double chain acid molecule form is transformed into the new determined nucleic acid fragment of strand containing non-sequenced fragments.2nd anchor(SEQ ID NO:102) with the hybridization articulated system of the second joint and process be: 28 DEG C, in the new determined nucleic acid fragment of strand, add 400 μ L 2 × SSPE, add the 2nd anchor(10 μM), 60 DEG C maintain 30s; Then 42 DEG C are cooled to, hybridization incubation 2min; After hybridization terminates, with magnet adsorption magnetic bead, 30 DEG C, 900 μ L cleaning buffer solutions clean, and are separated by unreacted 2nd anchor and remove.

5. obtain the sequence information of 6 Nucleotide after the 2nd anchor extending end.

Same procedure and the similar operations of 6 nucleotide sequence informations after an anchor extending end is obtained in refer step 2, connected by the fluorescent probe changing different mark position, read the nucleotide sequence information obtaining after the 2nd anchor extending end the 1 to 6.

6. obtain succeeding nucleotide sequence information.

The anchor more renewed, joint and restriction endonuclease, the operation of repeating step 3 to step 5, the nucleotide sequence read is excised by enzyme means of cutting, and build new determined nucleic acid fragment, the object extending forward order-checking is realized, until obtain the complete nucleotide sequence information that determined nucleic acid fragment can read with this.

The fluorescent signal view data that the determined nucleic acid fragment formed MCM6 gene nucleic acid segments and mthfr gene nucleic acid fragment checks order obtained, utilize and analyze with the Pstar II-Plus platform that checks order, obtain the nucleotide sequence that sequence number is respectively SEQ ID NO:105 and SEQ ID NO:106, more specifically analytical results is as shown in table 1.

Table 1. fluorescent signal analysis of image data result figure

Project	Front 100bases accuracy rate	100 ~ 200bases accuracy rate	200 ~ 300bases accuracy rate	The Average Accuracy of front 300bases
					MCM6 gene nucleic acid segments	99.98%（Q30）	99.88%（Q20）	99.26%（Q20）	99.74%（Q20）
Mthfr gene nucleic acid fragment	99.96%（Q30）	99.85%（Q20）	99.24%（Q20）	99.66%（Q20）

Wherein, Q30 described in table refers to read error rate in sequencing procedure and is only millesimal Nucleotide; Q20 refers to read error rate in sequencing procedure and is only centesimal Nucleotide.

According to table 1 data, in sequencing procedure, Q20 can reach 99% for trust data is as judging criterion, by the analytical data obtained in above-described embodiment, utilize the technical scheme described in the present invention carrying out MCM6, mthfr gene detection at the present embodiment, at least high-quality order-checking of 300bp can be reached and read long.

Simultaneously, in order to verify the accuracy of the present embodiment sequencing result, the mono-clonal plasmid sanger sequencing including MCM6 gene nucleic acid segments at first and include mthfr gene nucleic acid fragment is carried out sequencing, the result of the nucleotide sequence result obtained and this enforcement gained compared, comparison result shows: the sequencing result of the two is 100% in similarity.Therefore, the result that the present embodiment obtains is reliable result.

It should be noted that and carry out nucleotide sequence detection with the technical scheme described in the present invention, nucleotide sequence informations all for determined nucleic acid fragment should be read out in theory.But according to the data obtained in described embodiment, the high quality obtained in the present embodiment is read length and is at least 300bp, and along with reading long increase, it reads accuracy rate and presents progressively downward trend, its reason may be owing to cutting with attended operation at the enzyme of constantly circulation, and the concatenation ability that the enzyme of restriction endonuclease cuts ability and ligase enzyme declines to some extent and causes.Therefore, if the concatenation ability of cutting ability and ligase enzyme for the enzyme of restriction endonuclease is strengthened, utilize the technical scheme described in the present invention to detect nucleotide sequence, can reach longer read long.

Secondly, above-described embodiment is only a specific embodiments of technical scheme described in the present invention, for one or more gene orders in different testing sample, utilizes corresponding amplimer, the operation identical by above-described embodiment and step detect, and can obtain similar result equally.

In addition, the order-checking platform that the Treatment Analysis of sequencing procedure and fluorescent signal view data adopts can also be Pstar-II e, Pstar-II that Shenzhen HYK Gene Technology Co., Ltd. produces.

The source of testing sample of the present invention can be varied, and its source includes but not limited to blood, oral epithelium scrapes sampling, saliva, paraffin-embedded tissue and puncturing tissue etc.In testing process, when utilizing the technical scheme described in the present invention to detect gene order, kind and the number of the gene of detection are unrestricted, can arbitrary combination.Above-mentioned specific embodiment just detects a certain specific fragment in MCM6 and mthfr gene, if desired other regions of these two genes or some region of other genes are detected, only need design corresponding specificity amplification primer, corresponding specificity amplification primer is as described below to including but not limited to.

The primer of described ABCB1 is SEQ ID NO:1 and SEQ ID NO:2; The primer of described ADH2 is at least one pair of in SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; The primer of described ADH3 is at least one pair of in SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; The primer of described ALDH2 is at least one pair of in SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22; The primer of described APOB is SEQ ID NO:23 and SEQ ID NO:24; The primer of described APOE is at least one pair of in SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28; The primer of described CAT is SEQ ID NO:29 and SEQ ID NO:30; The primer of described CYBA is SEQ ID NO:31 and SEQ ID NO:32; The primer of described CYP1A1 is SEQ ID NO:33 and SEQ ID NO:34; The primer of described CYP2E1 is at least one pair of in SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42; The primer of described ERCC2 is at least one pair of in SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46; The primer of described LCT is at least one pair of in SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52; The primer of described LPL is at least one pair of in SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56; The primer of described MC1R is at least one pair of in SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60; The primer of described MCM6 is SEQ ID NO:61 and SEQ ID NO:62; The primer of described MTHFR is at least one pair of in SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66; The primer of described MTR is SEQ ID NO:67 and SEQ ID NO:68; The primer of described MTRR is SEQ ID NO:69 and SEQ ID NO:70; The primer of described NOS3 is SEQ ID NO:71 and SEQ ID NO:72; The primer of described OCA2 is SEQ ID NO:73 and SEQ ID NO:74; The primer of described PARP1 is SEQ ID NO:75 and SEQ ID NO:76; The primer of described PON1 is SEQ ID NO:77 and SEQ ID NO:78; The primer of described SOD3 is SEQ ID NO:79 and SEQ ID NO:80; The primer of described TYRP1 is SEQ ID NO:81 and SEQ ID NO:82; The primer of described VDR is at least one pair of in SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90; The primer of described XRCC1 is at least one pair of in SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94.

The primer of described CYP1A2 comprises SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, and at least one pair of in SEQ ID NO:111 and SEQ ID NO:112; The primer of described CYP3A4 comprises at least one pair of in SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118, SEQ ID NO:119 and SEQ ID NO:120, SEQ ID NO:121 and SEQ ID NO:122 and SEQ ID NO:123 and SEQ ID NO:124; The primer of described CYP3A5 comprises at least one pair of in SEQ ID NO:125 and SEQ ID NO:126 and SEQ ID NO:127 and SEQ ID NO:128; The primer of described CYP2C9 comprises at least one pair of in SEQ ID NO:129 and SEQ ID NO:130 and SEQ ID NO:131 and SEQ ID NO:132; The primer of described CYP2C19 comprises at least one pair of in SEQ ID NO:133 and SEQ ID NO:134 and SEQ ID NO:135 and SEQ ID NO:136; The primer of described CYP2D6 comprises at least one pair of in SEQ ID NO:137 and SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140, SEQ ID NO:141 and SEQ ID NO:142, SEQ ID NO:143 and SEQ ID NO:144, SEQ ID NO:145 and SEQ ID NO:146, SEQ ID NO:147 and SEQ ID NO:148 and SEQ ID NO:149 and SEQ ID NO:150; The primer of described ABCB1 comprises at least one pair of in SEQ ID NO:151 and SEQ ID NO:152, SEQ ID NO:153 and SEQ ID NO:154 and SEQ ID NO:155 and SEQ ID NO:156; The primer of described ADRB1 is SEQ ID NO:157 and SEQ ID NO:158; The primer of described ADRB2 is SEQ ID NO:159 and SEQ ID NO:160; The primer of described CACNA1C comprises at least one pair of in SEQ ID NO:161 and SEQ ID NO:162, SEQ ID NO:163 and SEQ ID NO:164 and SEQ ID NO:165 and SEQ ID NO:166; The primer of described NPPA is SEQ ID NO:167 and SEQ ID NO:168; The primer of described OPRM1 is SEQ ID NO:169 and SEQ ID NO:170; The primer of described VKORC1 is SEQ ID NO:171 and SEQ ID NO:172.

Detect MTHFR, FGFR2 gene in breast cancer susceptibility gene and IL-1 β gene, its specificity amplification primer sequence is respectively SEQ ID NO:173 and SEQ ID NO:174, SEQ ID NO:175 and SEQ ID NO:176, SEQ ID NO:177 and SEQ ID NO:178.

To the XRCC1 in lung cancer tumor susceptibility gene, APE1, CASP7, CASP8, CASP9, CHEK2, COX-2 and CYP1A1 gene carries out increasing and detecting, corresponding specificity amplification primer is SEQ ID NO:179 and SEQ ID NO:180, SEQ ID NO:181 and SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184, SEQ ID NO:185 and SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, SEQ ID NO:189 and SEQ ID NO:190, SEQ ID NO:191 and SEQ ID NO:192, SEQ ID NO:193 and SEQ ID NO:194.

Detect MMP2, SMAD7, ADH2, ALDH2, CYP1A2 gene in susceptibility gene of colorectal cancer, corresponding specificity amplification primer is SEQ ID NO:195 and SEQ ID NO:196, SEQ ID NO:197 and SEQ ID NO:198, SEQ ID NO:199 and SEQ ID NO:200, SEQ ID NO:201 and SEQ ID NO:202, SEQ ID NO:203 and SEQ ID NO:204.

Detect SNCA, LRRK2, PINK1, UCH-L1 and Parkin gene in parkinsonism tumor susceptibility gene, corresponding specificity amplification primer is SEQ ID NO:205 and SEQ ID NO:206, SEQ ID NO:207 and SEQ ID NO:208, SEQ ID NO:209 and SEQ ID NO:210, SEQ ID NO211 and SEQ ID NO:212, SEQ ID NO:213 and SEQ ID NO:214.

It should be noted that the present invention typically applies but is not limited to the detection of nutrition guide gene, conventional medicine genes involved, tumor susceptibility gene, in the gene test that other are similar, also can apply method set forth in the present invention.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

SEQUENCE LISTING

<110> Sheng department Tong

<120> mono-kind increases order-checking and reads long sequence measurement

<130>

<160> 214

<170> PatentIn version 3.3

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ctctttgcat gaaatgcttc cagg 24

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gaagccaatc ctggtgagta ga 22

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caggatgttg tgagtgagat taag 24

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gcccccatgt gtaatttatt gata 24

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cctggtgcct ggcttctagt a 21

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ggtggctgta ggaatctgtc a 21

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ggaaaaccaa ggcactgtaa t 21

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tcctacttac cctggttgaa tcta 24

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aagcactgta aaagcatatt gaag 24

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ggaaaaccaa ggcactgtaa t 21

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cctgggtggc ggcggctgac aag 23

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aggggaggac acgcagggtt caga 24

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ggggctccac aaacacacct cc 22

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gtgtgctata aacctggcct acc 23

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cctagcacct gaggaggtgt ag 22

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ctggagaaat ctgcttcccc 20

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gccacttcag ctgtctccct c 21

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ctggctgtga ggtggagatg act 23

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cagcagtgca tctagccatc tca 23

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tgggacgagg gcagagcaca tgt 23

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aagcaaaaac agaagaacaa caac 24

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gcatgtggtg gcactgccct c 21

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<211> 19

<212> DNA

<213> artificial sequence

<400> 93

ccacctgcca gcagcccac 19

<210> 94

<211> 19

<212> DNA

<213> artificial sequence

<400> 94

cagccccctc taccctcag 19

<210> 95

<211> 21

<212> DNA

<213> artificial sequence

<400> 95

gccggaagtc cgccacttca g 21

<210> 96

<211> 22

<212> DNA

<213> artificial sequence

<400> 96

ctgaagtggc ggacttccgg ct 22

<210> 97

<211> 27

<212> DNA

<213> artificial sequence

<400> 97

ttttttgccg gaagtccgcc acttcag 27

<210> 98

<211> 27

<212> DNA

<213> artificial sequence

<400> 98

ttttttctga agtggcggac ttccggc 27

<210> 99

<211> 20

<212> DNA

<213> artificial sequence

<400> 99

ctgaaguggc ggacutccgg 20

<210> 100

<211> 24

<212> DNA

<213> artificial sequence

<400> 100

gactgatagc ttcaggactg ctga 24

<210> 101

<211> 26

<212> DNA

<213> artificial sequence

<400> 101

tcagcagtcc tgaagctatc agtcnn 26

<210> 102

<211> 20

<212> DNA

<213> artificial sequence

<400> 102

cagucctgaa gctaucagtc 20

<210> 103

<211> 350

<212> DNA

<213> artificial sequence

<400> 103

atacgaccat ggaattcttc cctttaaaga gcttggtaag catttgagtg tagttgttag 60

acggagacga tcacgtcata gtttatagag tgcataaaga cgtaagttac catttaatac 120

ctttcattca ggaaaaatgt acttagaccc tacaatgtac tagtaggcct ctgcgctggc 180

aatacagata agataatgta gcccctggcc tcaaaggaac tctcctcctt aggttgcatt 240

tgtataatgt ttgattttta gattgttctt tgagccctgc attccacgag gataggtcag 300

tgggtattaa cgaggtaaaa ggggagtagt acgaaagggc attcaagcgt 350

<210> 104

<211> 349

<212> DNA

<213> artificial sequence

<400> 104

ccagtccctg tggtctcttc atccctcgcc ttgaacaggt ggaggccagc ctctcctgac 60

tgtcatccct attggcaggt taccccaaag gccaccccga agcagggagc tttgaggctg 120

acctgaagca cttgaaggag aaggtgtctg cgggagccga tttcatcatc acgcagcttt 180

tctttgaggc tgacacattc ttccgctttg tgaaggcatg caccgacatg ggcatcactt 240

gccccatcgt ccccgggatc tttcccatcc aggtgagggg cccaggagag cccataagct 300

ccctccaccc cactctcacc gcaccgtcct cgcacaggct gggggctct 349

<210> 105

<211> 315

<212> DNA

<213> artificial sequence

<400> 105

atacgaccat ggaattcttc cctttaaaga gcttggtaag catttgagtg tagttgttag 60

acggagacga tcacgtcata gtttatagag tgcataaaga cgtaagttac catttaatac 120

ctttcattca ggaaaaatgt acttagaccc tacaatgtac tagtaggcct ctgcgctggc 180

aatacagata agataatgta gcccctggcc tcaaaggaac tctcctcctt aggttgcatt 240

tgtataatgt ttgattttta gattgttctt tgagccctgc attccacgag gataggtcag 300

tgggtattaa cgagg 315

<210> 106

<211> 320

<212> DNA

<213> artificial sequence

<400> 106

ctcttcatcc ctcgccttga acaggtggag gccagcctct cctgactgtc atccctattg 60

gcaggttacc ccaaaggcca ccccgaagca gggagctttg aggctgacct gaagcacttg 120

aaggagaagg tgtctgcggg agccgatttc atcatcacgc agcttttctt tgaggctgac 180

acattcttcc gctttgtgaa ggcatgcacc gacatgggca tcacttgccc catcgtcccc 240

gggatctttc ccatccaggt gaggggccca ggagagccca taagctccct ccaccccact 300

ctcaccgcac cgtcctcgca 320

<210> 107

<211> 22

<212> DNA

<213> artificial sequence

<400> 107

cgggacttct tggatgctta tg 22

<210> 108

<211> 22

<212> DNA

<213> artificial sequence

<400> 108

aaaaaattag ctgggcgtga tg 22

<210> 109

<211> 20

<212> DNA

<213> artificial sequence

<400> 109

ccagccccag aagtggaaac 20

<210> 110

<211> 22

<212> DNA

<213> artificial sequence

<400> 110

actgatgcgt gttctgtgct tg 22

<210> 111

<211> 22

<212> DNA

<213> artificial sequence

<400> 111

tgaggcaaga ggattgtttg ag 22

<210> 112

<211> 23

<212> DNA

<213> artificial sequence

<400> 112

gaggacaagc cttaaattgg atg 23

<210> 113

<211> 21

<212> DNA

<213> artificial sequence

<400> 113

acaggcacac tccaggcata g 21

<210> 114

<211> 22

<212> DNA

<213> artificial sequence

<400> 114

cacacaccac tcactgacct cc 22

<210> 115

<211> 20

<212> DNA

<213> artificial sequence

<400> 115

tgtccccacc agattcattc 20

<210> 116

<211> 21

<212> DNA

<213> artificial sequence

<400> 116

tggagacctc cacaactgat g 21

<210> 117

<211> 18

<212> DNA

<213> artificial sequence

<400> 117

tccagaatag gcaaatcc 18

<210> 118

<211> 18

<212> DNA

<213> artificial sequence

<400> 118

caacaatcca caagaccc 18

<210> 119

<211> 18

<212> DNA

<213> artificial sequence

<400> 119

accgagtgga tttccttc 18

<210> 120

<211> 18

<212> DNA

<213> artificial sequence

<400> 120

tctggttacc tttgtggg 18

<210> 121

<211> 18

<212> DNA

<213> artificial sequence

<400> 121

tttgagggct tcacttag 18

<210> 122

<211> 18

<212> DNA

<213> artificial sequence

<400> 122

gcagtttctg ctggacat 18

<210> 123

<211> 18

<212> DNA

<213> artificial sequence

<400> 123

tggaagtgga cccagaaa 18

<210> 124

<211> 20

<212> DNA

<213> artificial sequence

<400> 124

cacccttaaa gatcacagat 20

<210> 125

<211> 23

<212> DNA

<213> artificial sequence

<400> 125

gatttacctg ccttcaattt ttc 23

<210> 126

<211> 23

<212> DNA

<213> artificial sequence

<400> 126

atccataccc ctagttgtac gac 23

<210> 127

<211> 21

<212> DNA

<213> artificial sequence

<400> 127

gaaggacggt aagaggtgct g 21

<210> 128

<211> 20

<212> DNA

<213> artificial sequence

<400> 128

gtgctctcca caaaggggtc 20

<210> 129

<211> 21

<212> DNA

<213> artificial sequence

<400> 129

gcaatggaaa gaaatggaag g 21

<210> 130

<211> 21

<212> DNA

<213> artificial sequence

<400> 130

cacccctgaa atgtttccaa g 21

<210> 131

<211> 20

<212> DNA

<213> artificial sequence

<400> 131

gagccacatg ccctacacag 20

<210> 132

<211> 20

<212> DNA

<213> artificial sequence

<400> 132

agccccaaac tggaaacaag 20

<210> 133

<211> 23

<212> DNA

<213> artificial sequence

<400> 133

ttgcttttaa gggaattcat agg 23

<210> 134

<211> 22

<212> DNA

<213> artificial sequence

<400> 134

aaatgtactt cagggcttgg tc 22

<210> 135

<211> 23

<212> DNA

<213> artificial sequence

<400> 135

taaattacaa ccagagcttg gca 23

<210> 136

<211> 22

<212> DNA

<213> artificial sequence

<400> 136

tgaatcacaa atacgcaagc ag 22

<210> 137

<211> 20

<212> DNA

<213> artificial sequence

<400> 137

gggtgtccca gcaaagttca 20

<210> 138

<211> 20

<212> DNA

<213> artificial sequence

<400> 138

cccgttctgt cccgagtatg 20

<210> 139

<211> 22

<212> DNA

<213> artificial sequence

<400> 139

catggagctc ttcctcttct tc 22

<210> 140

<211> 22

<212> DNA

<213> artificial sequence

<400> 140

catggagctc ttcctcttct tc 22

<210> 141

<211> 19

<212> DNA

<213> artificial sequence

<400> 141

gaatgctgtc cccgtcctc 19

<210> 142

<211> 19

<212> DNA

<213> artificial sequence

<400> 142

cagcctcccc tcattcctc 19

<210> 143

<211> 18

<212> DNA

<213> artificial sequence

<400> 143

tgggtgatgg gcagaagg 18

<210> 144

<211> 18

<212> DNA

<213> artificial sequence

<400> 144

ccagcagcct gaggaagc 18

<210> 145

<211> 19

<212> DNA

<213> artificial sequence

<400> 145

ggtggggcta atgccttca 19

<210> 146

<211> 19

<212> DNA

<213> artificial sequence

<400> 146

cgttgctcac ggctttgtc 19

<210> 147

<211> 18

<212> DNA

<213> artificial sequence

<400> 147

gcgtcccagg aggaatga 18

<210> 148

<211> 18

<212> DNA

<213> artificial sequence

<400> 148

cgggtgtccc agcaaagt 18

<210> 149

<211> 20

<212> DNA

<213> artificial sequence

<400> 149

tgtccagagg agcccatttg 20

<210> 150

<211> 21

<212> DNA

<213> artificial sequence

<400> 150

cctggtcgaa gcagtatggt g 21

<210> 151

<211> 22

<212> DNA

<213> artificial sequence

<400> 151

ctgagaacat tgcctatgga ga 22

<210> 152

<211> 20

<212> DNA

<213> artificial sequence

<400> 152

gctcccaggc tgtttatttg 20

<210> 153

<211> 23

<212> DNA

<213> artificial sequence

<400> 153

gcaggagttg ttgaaatgaa aat 23

<210> 154

<211> 22

<212> DNA

<213> artificial sequence

<400> 154

gtccaagaac tggctttgct ac 22

<210> 155

<211> 23

<212> DNA

<213> artificial sequence

<400> 155

tgaagttttt ttctcactcg tcc 23

<210> 156

<211> 21

<212> DNA

<213> artificial sequence

<400> 156

tctgtggggt catagagcct c 21

<210> 157

<211> 19

<212> DNA

<213> artificial sequence

<400> 157

ttcaactggc tgggctacg 19

<210> 158

<211> 18

<212> DNA

<213> artificial sequence

<400> 158

tccaggctcg agtcgctg 18

<210> 159

<211> 18

<212> DNA

<213> artificial sequence

<400> 159

gagcacgggc tggaactg 18

<210> 160

<211> 22

<212> DNA

<213> artificial sequence

<400> 160

aggacgatga gagacatgac ga 22

<210> 161

<211> 22

<212> DNA

<213> artificial sequence

<400> 161

aacataccca atgctctccc tc 22

<210> 162

<211> 22

<212> DNA

<213> artificial sequence

<400> 162

cacagtgaat taccacccca ag 22

<210> 163

<211> 18

<212> DNA

<213> artificial sequence

<400> 163

ggggaggaga gggcaact 18

<210> 164

<211> 18

<212> DNA

<213> artificial sequence

<400> 164

ggggaggaga gggcaact 18

<210> 165

<211> 21

<212> DNA

<213> artificial sequence

<400> 165

cgccaagtgt tcatctgtgt c 21

<210> 166

<211> 19

<212> DNA

<213> artificial sequence

<400> 166

ggcccaaacc tgaatctcc 19

<210> 167

<211> 21

<212> DNA

<213> artificial sequence

<400> 167

agaggcgagg aagtcaccat c 21

<210> 168

<211> 22

<212> DNA

<213> artificial sequence

<400> 168

gggaagcagg tggtcagtaa tc 22

<210> 169

<211> 19

<212> DNA

<213> artificial sequence

<400> 169

tctcggtgct cctggctac 19

<210> 170

<211> 20

<212> DNA

<213> artificial sequence

<400> 170

cgcacacgat ggagtagagg 20

<210> 171

<211> 20

<212> DNA

<213> artificial sequence

<400> 171

gggttcaagt ggttctcgtg 20

<210> 172

<211> 21

<212> DNA

<213> artificial sequence

<400> 172

tatcacagac gccagaggaa g 21

<210> 173

<211> 21

<212> DNA

<213> artificial sequence

<400> 173

cccagtccct gtggtctctt c 21

<210> 174

<211> 21

<212> DNA

<213> artificial sequence

<400> 174

actcagcgaa ctcagcactc c 21

<210> 175

<211> 21

<212> DNA

<213> artificial sequence

<400> 175

tcagaagttt ttgagagtgg c 21

<210> 176

<211> 21

<212> DNA

<213> artificial sequence

<400> 176

ctgtgatttg tatgtggtag c 21

<210> 177

<211> 21

<212> DNA

<213> artificial sequence

<400> 177

cagagagact cccttagcac c 21

<210> 178

<211> 21

<212> DNA

<213> artificial sequence

<400> 178

caatactctt ttcccctttc c 21

<210> 179

<211> 19

<212> DNA

<213> artificial sequence

<400> 179

cgcttctgtt gctaggctc 19

<210> 180

<211> 19

<212> DNA

<213> artificial sequence

<400> 180

tgtcaacgtc gtgggcttc 19

<210> 181

<211> 21

<212> DNA

<213> artificial sequence

<400> 181

gggacctgtc ttcctaactg c 21

<210> 182

<211> 20

<212> DNA

<213> artificial sequence

<400> 182

tcgggctgtt tatcgttgtg 20

<210> 183

<211> 21

<212> DNA

<213> artificial sequence

<400> 183

ttggtcgtct cctttctttc c 21

<210> 184

<211> 21

<212> DNA

<213> artificial sequence

<400> 184

cctggcaact ctgtcattca c 21

<210> 185

<211> 22

<212> DNA

<213> artificial sequence

<400> 185

ctactttatg aatgagccga gg 22

<210> 186

<211> 20

<212> DNA

<213> artificial sequence

<400> 186

caatgcttcc ttgaggtccc 20

<210> 187

<211> 20

<212> DNA

<213> artificial sequence

<400> 187

tgcgaactgg agtctgaggc 20

<210> 188

<211> 20

<212> DNA

<213> artificial sequence

<400> 188

tgtccccaga acctgccacc 20

<210> 189

<211> 21

<212> DNA

<213> artificial sequence

<400> 189

gcagatacaa actccaccct c 21

<210> 190

<211> 20

<212> DNA

<213> artificial sequence

<400> 190

gggttctaag ttccgctctc 20

<210> 191

<211> 21

<212> DNA

<213> artificial sequence

<400> 191

taaacactgt cacaagatgg c 21

<210> 192

<211> 22

<212> DNA

<213> artificial sequence

<400> 192

tacaggtgat tctaccctat ga 22

<210> 193

<211> 20

<212> DNA

<213> artificial sequence

<400> 193

cagcaggata gccaggaaga 20

<210> 194

<211> 20

<212> DNA

<213> artificial sequence

<400> 194

tccctctggt tacaggaagc 20

<210> 195

<211> 20

<212> DNA

<213> artificial sequence

<400> 195

aagcccactg agacccaagc 20

<210> 196

<211> 18

<212> DNA

<213> artificial sequence

<400> 196

gcacagggtg aggggatg 18

<210> 197

<211> 20

<212> DNA

<213> artificial sequence

<400> 197

gagacgcgta aaacttgctg 20

<210> 198

<211> 20

<212> DNA

<213> artificial sequence

<400> 198

gttctcaggt cagccttcca 20

<210> 199

<211> 22

<212> DNA

<213> artificial sequence

<400> 199

aggaatagta gggattagta gc 22

<210> 200

<211> 20

<212> DNA

<213> artificial sequence

<400> 200

tgtgcaagca ctttcgtctc 20

<210> 201

<211> 19

<212> DNA

<213> artificial sequence

<400> 201

gggagtgtaa cccataacc 19

<210> 202

<211> 19

<212> DNA

<213> artificial sequence

<400> 202

ccaccagcag accctcaag 19

<210> 203

<211> 20

<212> DNA

<213> artificial sequence

<400> 203

gagagccagc gttcatgttg 20

<210> 204

<211> 20

<212> DNA

<213> artificial sequence

<400> 204

ggctgagggt tgagatggag 20

<210> 205

<211> 22

<212> DNA

<213> artificial sequence

<400> 205

gctaaaaatg tctgctttgt cc 22

<210> 206

<211> 21

<212> DNA

<213> artificial sequence

<400> 206

tcatgaacaa gcaccaaact g 21

<210> 207

<211> 19

<212> DNA

<213> artificial sequence

<400> 207

tgggtgtttt gtgaggctg 19

<210> 208

<211> 22

<212> DNA

<213> artificial sequence

<400> 208

ttgcctactc caaggtttta tg 22

<210> 209

<211> 23

<212> DNA

<213> artificial sequence

<400> 209

aataatgaat gtcagtgcca gtg 23

<210> 210

<211> 22

<212> DNA

<213> artificial sequence

<400> 210

gtcctacagg gaaaatgctc tc 22

<210> 211

<211> 20

<212> DNA

<213> artificial sequence

<400> 211

tgctgccatc tgttctttgc 20

<210> 212

<211> 21

<212> DNA

<213> artificial sequence

<400> 212

catctctgac ctcgggaaaa c 21

<210> 213

<211> 21

<212> DNA

<213> artificial sequence

<400> 213

tgggaaaggt ttgatgctga t 21

<210> 214

<211> 19

<212> DNA

<213> artificial sequence

<400> 214

acgtccgtgg agggaagtg 19

Claims

1. a long sequence measurement is read in the increase order-checking for non-diseases diagnostic purpose, it is characterized in that, comprises the following steps:

Wherein, M, N are positive integer, 1≤M≤9,1≤N≤9; Described determined nucleic acid fragment contains nutrition guide gene order, conventional medicine related gene sequence or tumor susceptibility gene sequence;

Described nutrition guide gene refers to copying, transcribes, undergo mutation in accurate translation process after gene that the metabolism of nutritive substance is had an impact, described nutrition guide gene comprises at least one in ABCB1, ADH2, ADH3, ALDH2, APOB, APOE, CAT, CYBA, CYP1A1, CYP2E1, ERCC2, LCT, LPL, MC1R, MCM6, MTR, MTRR, NOS3, OCA2, PARP1, PON1, SOD3, TYRP1, VDR and XRCC1;

Described conventional medicine genes involved refers to encode to conventional medicine metabolism in vivo, release, intracellular signaling and receptor protein relevant gene or allelotrope, and described conventional medicine genes involved comprises at least one in CYP1A2, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2D6, ABCB1, ADRB1, ADRB2, CACNA1C, NPPA, OPRM1 and VKORC1;

Described tumor susceptibility gene refers to have certain association with human body specific characterization, and can disclose human body genetic constitution to the gene of healthy pros and cons and disease susceptibility situation or allelotrope, described tumor susceptibility gene comprises ACE, ADH2, AGT, ALDH2, ALOX5AP, APE1, APP, ApoE, ARL4C, AT1R, BICD1, CASP7, CASP8, CASP9, CDH1, CDNK2A/2B, CH25H, CHEK2, CHRNB2, COX-1, COX-2, CST3, CYP1A1, CYP1A2, CYP2E1, CYP11B2, DCC, DEPDC5, DNMT3B, eNOS, EGF, ERCC1, ERCC2, ERCC6, Exo1, FAM91A1, FasL, FGF, FGFR2, FOXQ1, GABBR1, GSTM1, GSTP1, GSTP1, G-β 3, HCG9, HLA-A, HLA-B/C, HLA-F, Hmlh1, hMLH1, HUMARA, IL-1 β, IL1-B, IL-2, IL-8, IL-10, ITGA9, KIF1B, LDLR, LL, LOC727677, LOC, LRRK2, LTF, MDM2, MDS1-EVI1, MEF2A, MICA, MMP-1, MMP2, MMP9, MTRR, MYO1D, NDFIP2, OGG1, p53, P73, Parkin, PARK2, PDGF, PINK1, PLCE1, PRPSAP2, PSCA, PS-1, PS-2, Rb, RnBO, RNF5P1, RNF43, SORL1, SMAD7, SNCA, TERT, TGFB1, TGF-β 1, TP53, TP63, TNFA, TNF-α, TNFRSF19, THSD7B, T-PA, UCH-L1, at least one in VEGF and XRCC1.

2. long sequence measurement is read in increase order-checking according to claim 1, and it is characterized in that, the fragment of determined nucleic acid described in steps A is fixed on surface of solid phase carriers.

3. long sequence measurement is read in increase order-checking according to claim 2, it is characterized in that, before steps A, also comprises step:

4. long sequence measurement is read in increase order-checking according to claim 3, and it is characterized in that, described steps A 0 comprises the following steps:

5. long sequence measurement is read in increase order-checking according to claim 4, it is characterized in that, primer described in steps A 02 comprises upstream primer for increasing to described source nucleic acid and/or downstream primer, described upstream primer holds the complementary nucleotide sequence combined, and described downstream primer is the nucleotide sequence identical with source nucleic acid 3 ' terminal sequence.

6. long sequence measurement is read in increase order-checking according to claim 4, it is characterized in that, the amplification described in steps A 03 is unit molecule amplification.

7. according to claim 4ly increase order-checking and read long sequence measurement, it is characterized in that, in described steps A 02, primer is incorporated into the mode of surface of solid phase carriers and is:

The group that primer and surface of solid phase carriers carry carries out pairing and is connected, and realizes directly combining;

Or carry out pairing by the group that the group that connexon carries carries with primer and surface of solid phase carriers respectively and be connected, realize indirectly combining.

8. long sequence measurement is read in increase order-checking according to claim 7, it is characterized in that, the mode that described pairing connects adopts at least one in biotin-avidin/Streptavidin, nanometer gold/iodacetyl-sulfydryl, amino-aldehyde radical/carboxyl/isothiocyano, acrylamide-silylation/polyacrylamide.

9. long sequence measurement is read in increase order-checking according to claim 1, and it is characterized in that, the first anchor primer described in steps A contains at least one enzyme and cuts recognition site.

10. long sequence measurement is read in increase order-checking according to claim 9, and it is characterized in that, described step C comprises the following steps:

11. increase order-checking according to claim 1 and read long sequence measurement, it is characterized in that, step C comprises the following steps:

Long sequence measurement is read in 12. increase order-checkings according to any one of claim 1 to 11, and it is characterized in that, described first anchor primer contains at least one specificity residue and/or one end is closed.

13. increase order-checking according to claim 12 and read long sequence measurement, it is characterized in that, described step B comprises the following steps: